METHODS AND COMPOSITIONS FOR THE TREATMENT OF LUNG DISEASES AND DISORDERS
A method of treating injured or diseased alveolar epithelial tissue in the lung of a mammal comprising transplanting into the lung a population of differentiated transgenic stem cells, or progeny thereof, which have an alveolar type II phenotype, effective to repair at least a portion of the injured or diseased alveolar epithelial tissue; an expression vector for transgenically modifying stem cells, comprising a DNA sequence encoding human surfactant protein C promoter operably linked to a DNA sequence encoding at least one drug-resistance gene; transgenic stem cells comprising such expression vector; and a method of preparing such transgenic stem cells and progeny thereof, a high percentage of which have an alveolar type II phenotype.
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This application is a continuation-in-part of U.S. patent application Ser. No. 12/527,969, filed Sep. 28, 2009, which is the 35 USC §371 U.S. National Stage of International Application No. PCT/US08/0545534 filed Feb. 21, 2008, which claims benefit of 60/890,958 filed Feb. 21, 2007. This application also claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/295,796 filed Jan. 18, 2010. The disclosures of those applications are hereby incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with U.S. Government support under Grant Nos. AI025011 and HL07433 awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELDThis disclosure relates to therapies for lung diseases and disorders and more particularly to methods and compositions for generating a culture of mammalian stem cell-derived alveolar type II (ATII) epithelial cells using a culture and genetic selection procedure that reliably yields numerous and sufficiently pure population of ATII cells. These methods generate stable transfected stem cell lines containing a single copy of a surfactant protein C (SPC)-promoter driven drug-resistant gene cassette. The stern cells are differentiated and cultured in the presence of the drug to produce sufficiently pure preparations of stern cell-derived ATII cells with biological and phenotypic characteristics of ATII cells, which include expression of surfactant proteins A, B, and C, presence of lamellar bodies, and the ability to proliferate and differentiate into ATI cells. These stem cell-derived ATII are sufficient in numbers and purity such that they can be used to treat and repair damaged lung tissue and improve lung function upon transplantation.
BACKGROUNDThe alveolar epithelium covers more than 99% of the internal surface area of the lung and is composed of two major cell types, the alveolar type I (ATI) epithelial cell and the alveolar type II (ATII) epithelial cell. ATI cells are large flat cells through which exchange of CO2/O2 takes place. They cover about 95% of the alveolar surface and comprise approximately 40% of the alveolar epithelium and 8% of the peripheral lung cells. In contrast, ATII cells are small, cuboidal cells, which cover about 5% of the alveolar surface and comprise 60% of the alveolar epithelium and 15% of the peripheral lung cells.
Traditionally the expression and synthesis of surfactant protein C (SPC) has been attributed to ATII cells, which also have a distinct morphological appearance of inclusion bodies, known as lamellar bodies. Important functions of ATII cells are: (i) to synthesize, store, and secrete surfactant, which reduces surface tension preventing collapse of the alveolus, (ii) to transport ions from the alveolar fluid into the interstitium, thereby minimizing alveolar fluid and maximizing gas exchange, (iii) to serve as progenitor cells for alveolar type I cells, which is particularly important during re-epithelialization of the alveolus after lung injury, and (iv) to provide pulmonary host defense by synthesizing and secreting several complement proteins including C3 and C5 (Strunk, et al., J Clin Invest 81, 1419-26, 1988; Rothman, et al., J Immunol 145, 592-8, 1990; Zhao, et al., Int J Mol Med 5, 415-9, 2000) as well as numerous cytokines and interleukins that modulate lymphocyte, macrophage and neutrophil functions (Mason, R. J. Respirology 11 Suppl, S 12-5, 2006).
Severe pulmonary diseases can be caused by deficiencies or genetic mutations in proteins synthesized by All cells that are important in maintaining normal lung homeostasis. For example, cystic fibrosis is caused by mutations in the transmembrane conductance receptor (CFTR) (Welsh, M. J., Ramsey, B. W., Accurso, F. J., Cutting, G. R. (2001) in The Metabolic Basis of Inherited Disease, ed. Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D. (McGraw-Hill, New York), pp. 5121-5188). CFTR is an important regulator of Cl− and liquid transport in the lung (Brochiero et al., Am J Physiol Lung Cell Mol Physiol 287, L382-92, 2004; Chroneos et al., J Immunol 165, 3941-50, 2000; Fang et al., Am J Physiol Lung Cell Mol Physiol 290, L242-9, 2006; Wang et al., Proc Natl Acad Sci USA 102, 186-91, 2005), and is functionally expressed by human ATII cells, strongly suggesting a critical role for CFTR in regulating ion and fluid transport in the lung alveolus (Fang et al., Am J Physiol Lung Cell Mol Physiol 290, L242-9, 2006; Wang et al., Proc Natl Acad Sci USA 102, 186-91, 2005). In addition, ATII cells synthesis and secrete the serine protease inhibitor, alpha-1 antitrypsin (α-1AT), which also plays a key role in alveolar homeostasis by regulating protease imbalance and adjusting fluid clearance (Swystun, et al., Am J Physiol Lung Cell Mol Physiol 288, L820-30, 2005; Boutten et al., Am J Respir Cell Mol Biol 18, 511-20, 1998), the importance of which is supported by the association of α-1AT deficiency with the development of pulmonary emphysema (Gadek et al., J Clin Invest 68, 889-98, 198112).
Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from stem cells provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Current treatments for lung injury do very little to assist cellular repair or to prevent the onset of lung fibrosis. Considerable interest has developed in the potential use of stem cells to repair lung epithelium destroyed by injury and disease. Initial investigations were performed using bone marrow derived stem cells, which at first were thought to engraft and differentiate into pulmonary epithelial cells following lung injury (Krause et al., Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105, 369-377, 2001; Theise et al., Radiation pneumonitis in mice: a severe injury model for pneumocyte engraftment from bone marrow. Exp Hematol 30, 1333-1338, 2002; Kotton et al., Bone marrow-derived cells as progenitors of lung alveolar epithelium. Development 128, 5181-5188, 2001; Grove et al., Marrow-derived cells as vehicles for delivery of gene therapy to pulmonary epithelium. Am J Respir Cell Mol Biol 27, 645-651, 2002; Ortiz et al., Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci USA 100, 8407-8411, 2003; Rojas et al., Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 33, 145-152, 2005). However, subsequent studies failed to reproduce these findings, revealing that the original conclusions drawn from rodent lung injury models were incorrect due to staining artifacts (Kotton et al., Failure of bone marrow to reconstitute lung epithelium. Am J Respir Cell Mol Biol 33, 328-334, 2005; Chang et al., Evidence that bone marrow cells do not contribute to the alveolar epithelium. Am J Respir Cell Mol Biol 33, 335-342, 2005). It is now generally agreed that following lung injury little, if any, actual physical attachment or engraftment of lung cells originating from the bone marrow occurred.
Although repair of the lung alveolar epithelium may include respiratory stem or progenitor cells not yet identified (Rawlins et al., Lung development and repair: contribution of the ciliated lineage. Proc Natl Acad Sci USA 104, 410-417, 2007; Kim, C. F. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823-835, 2005; Stripp, B. R. Hierarchical organization of lung progenitor cells: is there an adult lung tissue stem cell? Proc Am Thorac Soc 5, 695-698, 2008), it is well established that ATII cells have an important role in repopulating the injured alveolus by not only proliferating into new ATII cells but also by differentiating into ATI CELLs (Mason, R. J. Biology of alveolar type II cells. Respirology 11 Suppl, 12-15, 2006). Because ATII cells have numerous important functions, including the ability to proliferate and differentiate into the easily damaged ATI cell, stem cell-derived ATII cells are promising as a source of cells that could be used therapeutically to treat distal lung injury as well as pulmonary genetic disorders.
Embryonic stem (ES) cells are undifferentiated, pluripotent cells isolated from the inner cell mass of blastocyst-stage embryos are undifferentiated, pluripotent cells (Wobus, A. M. Mol Aspects Med 22: 149-64, 2001; Odorico, et al., Stem Cells 19: 193-204, 2001), which can be induced to differentiate in vitro into a wide range of different cell types (Wobus, et al., Differentiation 48: 173-82, 1991; Wobus, et al., J Mol Cell Cardiol 29: 1525-39, 1997; Muller et al., Faseb J 14: 2540-8, 2000; Okabe, et al., Mech Dev 59: 89-102, 1996; Brustle, et al., Science 285: 754-6, 1999; Kramer, et al., Mech Dev 92, 193-205, 2000; Buttery, et al., Tissue Eng 7, 89-99, 2001; Dani, et al., J Cell Sci 110 (Pt 11): 1279-85, 1997; Segev, et al., Stem Cells 22: 265-74, 2004). The potential clinical use of ES cells to regenerate or repair damaged tissue has fueled a tremendous amount of research activity to develop methods that promote the differentiation of ES cells into specific cell lineages. Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell. iPS cells appear similar to ES cells morphologically, and they have been shown to differentiate into cell types of all three germ layers in vitro as well as form teratomas in immune-deficient mice in vivo (Lengner C J. iPS cell technology in regenerative medicine. Ann. NY Acad. Sci. 1192, 38-44, 2010). iPS cells were first described after four transcription factors (Klf4, Sox2, Oct4, and c-Myc), where introduced into mouse fibroblasts through retroviral transduction, led to the formation of clones of cells with pluripotent properties (Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-76, 2006). Just one year later, human pluripotent cells were generated by either the same set of transcriptional factors or another set of transcriptional factors that did not include the oncogenic transgene c-Myc (Takahashi et al.,. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-72, 2007; Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-20, 2007). These reprogrammed pluripotent cells are now commonly referred to as induced pluripotent stem cells or iPS cells. The primary interest has been focused on the potential use of iPS cells in drug screening, modeling human diseases, and patient-specific therapies. Initially, iPS cells were made from somatic cells by retroviral or lentiviral transduction of the required transcription factors. Transgenes are inserted randomly by this method and may therefore disrupt normal gene expression. Recent efforts have focused on perfecting iPS technologies so that these pluripotent cells can be produced virus free and without insertional mutagenesis.
Experimentally primary ATII cells have shown promise in their ability to reverse lung injury in rats subjected to BLM challenge (Serrano-Mollar et al. Intratracheal transplantation of alveolar type II cells reverses bleomycin-induced lung fibrosis Am J Respir Crit Care Med 176, 1261-1268, 2007) indicating that endogenous lung cells with progenitor cell properties may prove useful in repairing damaged or diseased lung tissue. Recently, ES cells have been shown to differentiate into ATII cells in culture, but all of these procedures yield a mixture of ES cell derivatives with only a small percentage of the cells being ATII cells (Ali et al., Tissue Eng 8, 541-50, 2002; Samadikuchaksaraei et al., Tissue Eng 12, 867-75, 2006; Van Vranken et al., Tissue Eng 11, 1177-87, 2005; Rippon et al., Stem Cells 24, 1389-98, 2006). A mixed population of cells will not be suitable for transplantation, and remaining pluripotent stem cells in these mixed cultures carry a significant risk of producing teratomas after transplantation resulting in a possible lethal outcome (Wakitani, S. et al. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42, 162-165, 2003; Nussbaum, J. et al. Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. Faseb J 21, 1345-1357, 2007). Therefore, a major prerequisite for using ATII cells therapeutically in humans is to achieve a pure population of sufficient stem cell derived-ATII cells. Prior to the disclosed methods, routinely obtaining sufficient quantities and a pure population of ATII cells to allow for clinical treatment of lung disease was not been possible. The present disclosure also describes the therapeutic activity of stem cell derived-ATII cells derived from human stem cells as a treatment of lung injury.
SUMMARYDisclosed are compositions and methods that can be used to reliably generate a culture of stem cell-derived ATII cells using a culture and genetic selection procedure that reliably yields an essentially pure population of mammalian (human) ATII cells. These methods are used to generate stable transfected stem cell lines containing a single copy of a SPC-promoter driven drug-resistant gene cassette. When used with mammalian, such as, human embryonic stem cells (hES) the process does not require the formation of embryoid bodies when embryonic stem cells are used. The stem cells are differentiated and cultured in the presence of the drug to produce sufficiently pure preparations of stem cell-derived ATII cells with biological and phenotypic characteristics of ATII cells, which include expression of surfactant proteins A, B, and C, presence of lamellar bodies, and the ability to proliferate and differentiate into ATI cells. These stem cell-derived ATII cells are sufficient in numbers and purity such that they can be used to treat and repair damaged lung tissue upon transplantation. Transplantation of the human stem cell derived-ATII cells after lung injury greatly reduced the extent of damage within the lung, as evidenced by the presence of only a few isolated, small areas of injured tissue surrounded by much larger areas of normal alveolar structure. This therapy also improved lung function, as evidenced by the reversal of the body weight loss that accompanies acute lung injury and a reversal of injury impaired lung tidal volumes and blood arterial oxygen saturation levels to normal levels.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and/or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.
As used herein, and unless otherwise indicated, the terms “treat”, “treating”, and “treatment” contemplate an action that occurs while a patient is suffering from lung injury or disorders that reduces the severity of one or more symptoms or effects of the lung injury or disorder, or a related disease or disorder. Where the context allows, the terms “treat”, “treating”, and “treatment” also refers to actions taken toward ensuring that individuals at increased risk of lung injury or disorder are able to receive appropriate surgical and/or other medical intervention prior to onset of lung injury or lung disorder. As used herein, and unless otherwise indicated, the terms “prevent”, “preventing”, and “prevention” contemplate an action that occurs before a patient begins to suffer from lung injury or disorder that delays the onset of, and/or inhibits or reduces the severity of, a lung injury or disorder. As used herein, and unless otherwise indicated, the terms “manage”, “managing”, and “management” encompass preventing, delaying, or reducing the severity of a recurrence of lung injury or disorder in a patient who has already suffered from such a disease or condition. The terms encompass modulating the threshold, development, and/or duration of the lung injury or disorder or changing how a patient responds to the lung injury or disorder.
As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound is an amount sufficient to provide any therapeutic benefit in the treatment or management of lung injury or disorder or to delay or minimize one or more symptoms associated with lung disorders. A therapeutically effective amount of a compound means an amount of the compound, alone or in combination with one or more other therapies and/or therapeutic agents that provides any therapeutic benefit in the treatment or management of lung injuries or disorders, or related diseases or disorders. The term “therapeutically effective amount” can encompass an amount that alleviates lung injuries or disorders, improves or reduces lung injury or disorder, improves overall therapy, or enhances the therapeutic efficacy of another therapeutic agent.
As used herein, and unless otherwise specified, a “prophylactically effective amount” of a compound is an amount sufficient to prevent or delay the onset of lung injury or disorder, or one or more symptoms associated with lung injury or disorder or prevent or delay its recurrence. A prophylactically effective amount of a compound means an amount of the compound, alone or in combination with one or more other treatment and/or prophylactic agent that provides a prophylactic benefit in the prevention of lung injury or disorder. The term “prophylactically effective amount” can encompass an amount that prevents lung injury or disorder, improves overall prophylaxis, or enhances the prophylactic efficacy of another prophylactic agent.
Introduction.Respiratory diseases are a major cause of mortality and morbidity worldwide and current treatments offer no prospect of cure or disease reversal. Current therapies rely on management of symptoms or lung transplantation. Unfortunately, patients requiring lung replacement will often succumb to their disease owing to the scarcity of donated lung tissue suitable for transplantation. There is, therefore, a pressing need to develop novel therapeutic strategies to treat lung disease.
Lung injury and lung disorders, include but are not limited those that are acute or chronic. Acute lung injury and Acute Respiratory Distress Syndrome (ARDS) are syndromes of widespread lung inflammation and increased pulmonary vascular permeability. Acute lung injury and ARDS are characterized by a diffuse heterogeneous lung injury characterized by hypoxemia, non cardiogenic pulmonary edema, low lung compliance and widespread capillary leakage. Acute lung injury and ARDS are essentially the same clinical disorder and differ only in degree. Acute lung injury and ARDS are caused by any stimulus of local or systemic inflammation. The exact incidence of Acute lung injury and ARDS is uncertain. However, recent estimates indicate that about 190,000 cases/yr occur in the US alone.
Acute lung injury and ARDS can be caused by direct lung injury or indirect lung injury. Causes of direct (or primary) lung injury include, but are not limited to, aspiration, pneumonia, diffuse alveolar hemorrhage, fat embolism, lung transplantation, near-drowning, pulmonary contusion, toxic gas inhalation and inhalation of hot gasses. For example, inhalation of smoke and toxic fumes from chemicals like chlorine can lead to diffuse bronchiolitis and chronic respiratory sequelae including decreased lung function and persistence of asthma.
Causes of indirect (or secondary) lung injury include, but are not limited to, sepsis, severe trauma, bone marrow transplantation, burns, cardiopulmonary bypass, drug overdose (e.g., aspirin, cocaine, opioids, phenothiazines, tricyclics), massive blood transfusion and pancreatitis
Because acute lung injury and ARDS are syndromes of widespread lung inflammation, it follows that lung disorders or syndromes that lead to inflammation and lung injury may also benefit from the described methods. Additional disorders that are associated with lung inflammation and injury include, but are not limited to, cystic fibrosis and those that result from: infections due to viruses, bacteria (tuberculosis), parasites, fungi, mycoplasma; immune or connective tissue diseases or disorders (such as but not limited to systemic lupus erythematosus, rheumatoid arthritis, asthma, COPD); organ failure (pancreatitis, liver failure, cirrhosis, kidney or renal failure—uremia, nephritic syndrome); cancer (such as but not limited to breast cancer, bronchogenic carcinoma, metastatic lung cancer, tumors that are found in the pleura, heart failure); chemical exposure (for example, asbestos); lung infarction and pulmonary emboli (blood clots in a vessel that goes to the lungs); obstruction of lymph channels; and trauma. The most distal region of the lung is a complex organization of alveoli where O2/CO2 exchange occurs.
The alveolar epithelium is composed of two types of epithelial cells, type I (ATI cells) and type II (ATII cells). The ATI cells are large yet highly flattened cells with multiple apical surfaces that extend into adjacent alveoli. These cells, together with the endothelium of the surrounding capillaries, form the very thin blood-air interface that is essential for O2/CO2 exchange. In contrast, ATII cells are small cuboidal cells that secrete surfactant, which reduces surface tension, preventing collapse of the alveolus. The lung is constantly exposed to environmental toxins and pathogens that can destroy alveolar epithelial cells, in particular the thin injury prone ATI cells. The ability of the injured alveolar epithelium to quickly and efficiently self-repair is therefore very important for maintaining normal pulmonary function. Although repair of the lung alveolar epithelium may include respiratory stem or progenitor cells not yet identified, it is well established that ATII cells have an important role in repopulating the injured alveolus by not only proliferating into new ATII cells but also by differentiating into ATI cells.
Despite the endogenous repair capacity of the alveolar epithelium, it is often not sufficient. Inadequate, delayed, or impaired re-epithelialization of the injured alveolus is regarded as a key factor in the pathogenesis of several life-threatening pulmonary diseases, including acute lung injury, acute respiratory distress syndrome, and chronic obstructive pulmonary disorder. Current treatments for lung alveolar epithelial injury at best provide symptomatic relief but offer no prospect to repair the damaged epithelium or prevent lung fibrosis. Consequently, there is a pressing need for the development of novel therapies that facilitate the regeneration of alveolar epithelium destroyed by acute and chronic lung diseases, which also include, but are not limited to genetic disorders that result from inherited mutations (such as, but not limited to, cystic fibrosis, alpha-1-antitrypsin deficiency, and surfactant protein deficiencies).
Embryonic stem (ES) cells are self-renewing pluripotent cells, which can be induced to differentiate into a wide range of different cell types. The potential use of ES cells in the treatment of pulmonary diseases has evoked extensive interest in developing methods that promote ES cell differentiation into lung progenitor cells. Because of their ability to proliferate and to differentiate into ATI cells, ATII cells derived from ES cells are thought to be promising as a transplantable source of cells that will be useful therapeutically to treat distal lung injury. Recently, ES cells were shown to differentiate into ATII cells in culture, but these procedures yielded a mixture of ES cell derivatives with only a small percentage of the cells being ATII cells. A mixed population of cells will not be suitable for transplantation, and remaining pluripotent cells in these mixed cultures carry a significant risk of producing teratomas after transplantation. Therefore, a prerequisite for using ATII cells therapeutically is to develop a method that will routinely yield a pure population of ATII cells. The present inventors have recently achieved this goal by generating stable human ES cell lines that can be differentiated and enriched into a pure population of ATII cells (as described in co-pending U.S. patent application Ser. No. 12/527,969 filed Aug. 20, 2009, the disclosure of which is hereby incorporated herein by reference).
The disclosed compositions and methods can be used to reliably generate a culture of stem cell-derived ATII cells using a culture and genetic selection procedure that reliably yields an essentially pure population of mammalian (such as but not limited to human) ATII cells. These methods generate stable transfected stem cell lines containing a single copy of a SPC-promoter driven drug-resistant gene cassette. When used with mammalian embryonic stem cells (hES) the process does not require the formation of embryoid bodies when embryonic stem cells are used. The stem cells are differentiated and cultured in the presence of the drug to produce sufficiently pure preparations of stem cell-derived ATII cells with biological and phenotypic characteristics of ATII cells, which include expression of surfactant proteins A, B, and C, presence of lamellar bodies, and the ability to proliferate and differentiate into ATI cells. These stem cell-derived ATII are sufficient in numbers and purity such that they can be used to treat and repair damaged lung tissue upon transplantation. Transplantation of the human stem cell derived-ATII cells after lung injury (as exemplified in mice that were treated with and without hES-ATII cells following BLM-induced acute lung injury) greatly reduced the extent of damage within the lung, as evidenced by the presence of only a few isolated, small areas of injured tissue surrounded by much larger areas of normal alveolar structure. This therapy also improved lung function, as evidenced by the reversal of the body weight loss that accompanies acute lung injury and a reversal of injury impaired lung tidal volumes and blood arterial oxygen saturation levels to normal levels.
To circumvent the inherent difficulties in generating a pure culture of ES cell-derived ATII cells, a culture and genetic selection procedure that reliably yields an essentially pure population of human ATII cells by generating stable transfected human embryonic stem (hES) cell lines containing a single copy of a SPC-promoter driven drug resistance (for example, neomycin-resistance) gene cassette. Without having to form embryoid bodies, these hES cell lines can be differentiated and cultured in the presence of neomycin to produce pure preparations of hES cell-derived ATII cells with biological and phenotypic characteristics of ATII cells as well as the ability to proliferate and differentiate into ATI cells
In some embodiments, a culture method that efficiently induces direct differentiation of ES cells into ATII cells without EB formation, and also a method of producing a highly pure population of ATII cells. In some embodiments the method results in a clonal population of ATII phenotype cells sufficiently pure (e.g., at least 99% ATII phenotype) to be suitable for implantation into a mammalian host lung tissue without significant risk of producing teratomas.
The exemplary stem cell differentiated ATII cells appear morphologically normal, express the characteristic surfactant proteins A, B, and C, CFTR and α-1AT RNA as well as synthesize and secrete complement proteins C3 and C5. Thus, a unique approach is provided to reliably generate significant quantities of sufficiently pure hES cell-derived ATII cells that will potentially be used in the future to reconstitute damaged lung alveolus and other lung diseases or disorders such as, but not limited to, genetic diseases that affect the lung.
Embodiments of the present methods result in direct differentiation of ES cells into ATII cells which contrasts with previous attempts at differentiation of ATII cells from hES cells, in which multiple steps were used to derive ATII cells from hES cells through EB formation. Previous approaches require prolonged time periods to develop the endoderm from which the ATII cells are derived, and yet in the end the produce scarcely detectable numbers of ATII cells in mixed cell populations. Therefore, in addition to providing sufficiently pure and numerous ATII cells, embodiments of the present methods decrease the time and effort in generating hES-derived ATII cells and facilitate their therapeutic and clinical use.
As documented herein by RT-PCR, flow cytometric analysis, and immunostaining, using hES cells (stem cells) cultured on matrix gel coated dishes, it is demonstrated that hES cells did in fact differentiate directly into ATII cells without embryonic body (EB) formation. In addition, SPC expression indicating the presence of ATII cells in the differentiating hES cell cultures occurred 5 days sooner in the absence of EB formation. Moreover, 11.2% of differentiated cells cultured on the matrix gel coated dishes were determined to express SPC protein on day 15 compared to 2.8% on day 33 when the EB formation approach was employed. It is proposed, in view of these collective results, that the components of matrix gel, such as laminin and collagen IV, may not only efficiently maintain the biological characteristics of ATII cells but also encourage differentiation of hES cells to ATII cells.
The use of ES cells as a source of transplantable cells in the lung alveolus will require the generation of significant quantities of highly pure ATII cells. To achieve this goal, genetic modification of hES cells was chosen so that the resulting differentiated ATII cells could be enriched through antibiotic selection. This approach was to establish a stable transfected hES cell line containing a single copy of the human SPC promoter-Nedr fusion gene. When subjected to differentiation in vitro, it was hypothesized that ATII cells derived from this genetically modified hES cell line (SPCP/NEO.74) would express the Nedr gene and would therefore survive G418 antibiotic selection, whereas, all the other differentiated cell lineages as well as the pluripotent cells would be eliminated by G418 selection. Immunocytochemical and flow cytometric analysis of the surviving G418 selected cells supported this hypothesis, indicating that this genetic selection approach resulted in an enrichment of hES-ATII cells to more than 99% when cultured on matrix gel coated plates. The above-described protocol reproducibly produced from each 10 cm culture dish more than 106 essentially pure ATII cells within 15 days of differentiation, which will provide in a timely manner sufficient numbers of pure ATII cells for future transplantation investigations.
Ultrastructural examination by transmission electron microscopy and Papanicolaous staining demonstrated that the hES-ATII cells are morphologically normal and exhibit typical Lamellar bodies, which are a characteristic hallmark of primary ATII cells. The hES-ATII cells were shown to exhibit normal important biological functions, such as the synthesis of surfactant proteins A, B, and C. Moreover, these cells expressed RNA specific for CFTR and α-1AT, suggesting that they may have therapeutic value in the treatment of patients with cystic fibrosis or α-1AT deficiency. The hES-ATII cells also synthesized and secreted complement proteins C3 and C5, which are important in inflammation and host defense in the lung. Activation of C3 and C5 produces the potent complement anaphylatoxins, C3a and C5a. Recent reports indicate that C3a and C5a have novel and important roles in tissue regeneration, and neurogenesis. It is proposed in light of these findings that C3 and C5 synthesized and secreted by ATII cells are not only important in mediating pulmonary inflammation and host defense but could also play critical biological roles in alveolus regeneration and repair. In conclusion, this study provides the first description of a reliable single step procedure that can be employed to drive the differentiation of hES cells into a highly pure population of ATII cells, thereby providing a practical source of cells for repair of distal lung injury and for potential treatment of pulmonary genetic disorders.
In some embodiments, production of a population of in vitro cultured cells of alveolar epithelial type II (ATII) cell lineage derived from at least one embryonic stem cell includes: (a) culturing at least one embryonic stem cell in vitro in a suitable medium (e.g., Matrigel®), to produce differentiated cells without formation of an embryonic body. At least some of the differentiated cells are of ATII cell phenotype. The method of production further includes (b) identifying the differentiated cells of ATII cell phenotype by detecting expression of at least one biomarker of ATII cells, and (c) isolating the differentiated cells having ATII cell phenotype. In some cases, this may include selecting a purified population of differentiated cells wherein at least 95%, and in some cases at least 99%, of the cells have ATII cell phenotype. The method further includes (d) cloning the isolated cells to produce a population of cells having ATII cell phenotype. For some application, this includes producing a population of more than 106 cells within 15 days of differentiation, wherein at least 95%, and in many cases at least 99%, of the cloned cell population have ATII phenotype.
In some instances the selected biomarker comprises the surfactant protein C (SPC). In some instances the selected biomarker comprises cystic fibrosis transmembrane conductance receptor (CFTR). In some instances the selected biomarker comprises α-1 -antitrypsin (α-1 AT). In still other instances the selected biomarker comprises complement protein C3 or C5, or both.
In some embodiments, a construct comprises a DNA sequence encoding human surfactant protein C promoter operably linked to a DNA sequence encoding at least one drug-resistance gene. In some embodiments, drug-resistance gene is a neomycin-resistance gene. In some embodiments, a linear expression vector comprises a construct as described herein operably linked downstream of a DNA sequence encoding a 3′-hprt vector, wherein the 3′-hprt vector comprises a puromycin-resistance gene.
In some embodiments, a transgenic stem cell comprises an above-described expression vector. A stem cell employed in some of the disclosed methods comprises a transgene operably linked to a cell-specific promoter. For example, the transgene may comprise a drug resistance gene that, when expressed, is capable of imparting resistance to the drug in the stem cell or progeny thereof.
In some embodiments, a disclosed transgenic stem cell further contains an expressible therapeutic transgene operably linked to a cell-specific promoter. In some embodiments, a disclosed transgenic stem cell is a transgenic induced pluripotent stem cell.
An in vivo method of repairing injured or diseased alveolar epithelial tissue in the lung of a mammal is provided in accordance with some embodiments. Such method comprises transplanting into a lung that contains injured or diseased alveolar epithelial tissue, a population of differentiated stem cells, or progeny thereof, at least 95% of which have ATII phenotype. The population of cells is prepared in accordance with a method described herein, and is effective to repair at least a portion of the injured or diseased alveolar epithelial tissue. In some embodiments, the mammal suffers from a genetic disease affecting alveolar epithelial tissue in the lung, and said therapeutic transgene encodes a gene product for ameliorating the detrimental effects of said genetic disease in said alveolar epithelial tissue. In some embodiments, at least one differentiated stem cell, or progeny thereof, comprises a therapeutic transgene operably linked to a cell-specific promoter, wherein the transgene encodes a therapeutic gene product. In some embodiments, an above-described population of cells is transplanted directly to injured or diseased alveolar epithelial tissue in said lung. In some embodiments, transplanting the population of cells comprises administering them into the lung endotracheally via oropharynx intubation.
Advances in stem cell research during the past decade have fueled a tremendous amount of excitement over the possibility of using adult or embryonic sources of stem cells to repair or regenerate damaged organs. The complex structure of the lung, the plethora of different cell types comprising the lung parenchyma, and an incomplete understanding of endogenous lung repair at a molecular and cellular level have made the development of stem cell therapeutics for pulmonary medicine appear rather daunting.
Despite these challenges, significant progress has been achieved during the past several years in identifying early progenitor lung cells, developing procedures for the generation and purification of these progenitor cells from embryonic stem (ES) and induced pluripotent stem (iPS) cell sources, evaluating the therapeutic potential of these cells in animal models of lung disease, and their application in lung tissue regeneration using bioengineered three-dimensional matrices and scaffolding.
Secretory protein C (SPC), is considered a protein restricted in expression to alveolar type II cells. In the gas-exchanging region of the lung, alveolar type II epithelial (ATII) cells have been traditionally considered the progenitor cell of the alveolar epithelium, based on data published by numerous laboratories. Collectively, studies have shown that ATII cells exhibit biological hallmarks of progenitor cells, including the ability to proliferate as well as differentiate into other cell types (ATI cells). Moreover, animal models of lung injury have shown that ATII cells are critical in repopulating the injured alveolus, not only by generating new ATII cells but also by differentiating into and replacing the thin, injury-prone ATI cells. It remains uncertain if all ATII cells or only a putative subpopulation of ATII cells act as progenitor cells for the alveolar epithelium.
However, the repair capacity provided by endogenous lung epithelial progenitor cells is often insufficient. Inadequate, delayed, or impaired re-epithelialization of the injured lung is regarded as a key factor in the pathogenesis of several life-threatening pulmonary diseases, such as, but not limited to, chronic obstructive pulmonary disease (COPD). Moreover, the natural repair capacity provided by endogenous epithelial progenitor cells appears to diminish with age.
The repair process provided by the lung epithelial progenitor cells could be greatly enhanced or restored by the introduction of endogenous epithelial progenitor cells to debilitated lungs, reversing the effects of injury, disease, or aging. Although this approach is promising in theory, it has proven very difficult to obtain sufficient numbers of primary lung epithelial progenitor cells to be used effectively and routinely in a clinical setting.
Recent attempts to resolve this problem have focused on generating lung epithelial progenitor cells from differentiated cultures of embryonic stem cells. Moreover, with the recent discovery that iPS cells can be created from dermal skin fibroblasts, investigations have begun to examine the possibility of generating lung progenitor epithelial cells from somatic cells. If these efforts succeed, patient-specific iPS cells could be obtained, thereby avoiding the immune rejection problems that might occur if heterologous sources of embryonic stem cells were employed. Moreover, iPS cells offer the possibility of generating gene-corrected, patient-specific lung progenitor cells from individuals with genetic diseases affecting the lung, including cystic fibrosis, alpha-1-antitrypsin deficiency, and surfactant protein deficiencies.
Embryonic stem (ES) cells are undifferentiated, pluripotent cells. Under well-defined culture conditions, ES cells can be maintained indefinitely as rapidly proliferating undifferentiated cells. In their undifferentiated state, they maintain the ability to give rise to cells of all three embryonic germ layers (ectodermal, mesodermal, and endodermal). The potential clinical use of ES cell-derived progenitor cells to regenerate or repair damaged tissue has fueled a tremendous amount of excitement and research activity in numerous medical disciplines. However, despite the plethora of documented research efforts devoted to other tissues, such as the heart and spinal cord, research publications examining the use of ES cells for pulmonary regenerative medicine have remained scarce. For one thing there are numerous political and financial issues that partly explain the lack of ES cell research in pulmonary medicine. Another factor that has slowed research efforts is the finding that endodermal cells, such as lung epithelial cells, are much more difficult to generate from undifferentiated ES cell cultures than are mesodermal and ectodermal cells (Rippon et al., Embryonic stem cells as a source of pulmonary epithelium in vitro and in vivo. Proc. Am. Thorac. Soc. 5:717-22, 2008). During embryonic development, the endoderm is the last of the three germ layers to form, and its cellular specification process is complex. As a consequence, the development of differentiation and purification procedures for ES cell-derived lung epithelial cells, compared to cardiomyocytes and neuronal cells, has proven to be especially difficult.
Furthermore the generation of ATII cells from differentiated ES cell cultures has continued to be problematic, with the percentage of ATII cells in the differentiated cultures remaining a minority of the overall cell content. Improvements to the generation of ATII cells from ES cell cultures has been achieved by the use of conditioned media from a human ATII tumor cell line (A549) in combination with compounds known to drive endodermal cell development (Roszell et al., Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application. Tissue Eng. Part A 15:3351-65, 2009); however, these procedures did not achieve the level of purity that would be required for clinical use. Moreover, these mixed cell cultures may contain undifferentiated pluripotent cells, which could develop into teratomas after transplantation.
Some embodiments of the present compositions and methods utilize a culture and genetic selection procedure that reliably yields an essentially pure population of human ATII cells by generating stable transfected human stem cell lines containing a single copy of a SPC-promoter driven neomycin-resistant gene cassette.
Many embodiments of the present compositions and methods efficiently induce direct differentiation of human stem cells into ATII cells without EB formation and also produce a highly pure population of human ATII cells. In some embodiments the method results in a clonal population of ATII phenotype cells sufficiently pure (e.g., at least 95% and in many cases at least 99% ATII phenotype) suitable for implantation into a mammalian host lung tissue without significant risk of producing a teratoma.
The human stem cell lines can be differentiated and cultured in the presence of neomycin to produce pure preparations of human stem cell-derived ATII cells with biological and phenotypic characteristics of ATII cells, including expression of surfactant proteins A, B, and C, presence of lamellar bodies, and the ability to proliferate and differentiate into ATI cells (as further described in the Examples below).
Failure to efficiently repopulate the alveolar epithelium after injury is a major reason for the development of pulmonary fibrosis. Alveolar epithelial cell damage results in denuded epithelial basement membranes, release of inflammatory and chemoattractant molecules (which facilitate the migration of fibroblasts into the alveolar space), and development of intra-alveolar fibrosis.
In some embodiments of the described methods, human stem cell lines can be differentiated and cultured in the presence of neomycin to produce pure preparations of human stem cell-derived ATII cells, which when transplanted (via oropharynx intubation) into the lungs of SCID mice subjected to bleomycin induced acute lung injury, a significant number of stem cell derived ATII cells prepared by the methods described physically engraft into the alveolar epithelial basement membranes denuded by bleomycin injury. By the end of the study (10 days post injury), several of these engrafted cells expressed ATI phenotypic cell markers, suggesting that they had differentiated or were in the process of differentiating into ATI cells.
Transplantation of the human stem cell-derived ATII cells, produced using embodiments of the described methods reversed or prevented acute lung injury in the bleomycin-treated mice when transplanted 1 or 2 days following injury, as demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Interestingly, some injured lung alveolar epithelium regions appeared healthy after transplantation of the human stem cell-derived ATII cells, produced using embodiments of the described methods, despite having no observable engrafted human stem cell-derived ATII cells. This suggests that human stem cell-derived ATII cells, produced using embodiments of the described methods, may provide paracrine repair/protection to injured lung epithelium, such as, but not limited to, the release of anti-inflammatory mediators such as IL-10, angiopoietin-1, and keratinocyte growth factor.
There are no known surface protein markers that could be exploited for the purification (for example, by flow or magnetic beads) of lung progenitor cells, thus one of the potential advantages that use of many embodiments of the described methods provides is the opportunity to provide a pure and numerous population of cells that function like ATII cells. Because of their robust pluripotent nature, hES and iPS cells exhibit more therapeutic potential repair and regeneration of injured lung epithelium than adult bone marrow-derived stem cells for modifications such as those disclosed in embodiments of the described methods. However, However, the direct administration of hES or iPS cells that may still be pluripotent could pose a risk of teratoma formation, especially to patients who are immunocompromised. A potential advantage of many embodiments of the disclosed methods is that they provide a mechanism by which stem cells that have developed to form ATII cells can be specifically selected and purified.
In some embodiments, the alveolar type II epithelial cells derived from human stem cells (such as but not limited to hES cells, as used in the examples) is used therapeutically in the treatment of lung injury. Therapeutic activity is demonstrated herein using alveolar type II epithelial cells derived from hES cells (hES-ATII cells) in an animal model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin-induced acute lung injury, hES-ATII cells behaved as normal primary ATII cells, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATII cells, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATII cells shows promise as an effective therapeutic to treat acute lung injury and related disorders.
As described in some embodiments, is the therapeutic activity of alveolar type II epithelial cells derived from human ES cells (hES-ATII cells) in the treatment of lung injury. This activity is demonstrated using alveolar type II epithelial cells derived from hES cells (hES-ATII cells) in an animal model of acute lung injury. Provided is evidence that transplanted human ES cells derived ATII cells (hES-ATII cells) will differentiate and functionally repair of epithelium of the acutely injured alveolus in a mouse model of bleomycin (BLM)-induced acute lung injury.
Also described in some embodiments is the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATII cells) in an animal model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin-induced acute lung injury, hES-ATII cells behaved as normal primary ATII cells, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATII cells, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATII cells shows promise as an effective therapeutic to treat acute lung injury
Because of their ability to differentiate into essentially any cell in the body, ESCs may provide an attractive alternative to bone marrow stem cells in regenerating lung tissue. However, much of the enthusiasm for using hESCs to regenerate damaged tissue has been tempered by the observation that direct application of hESCs in vivo will likely cause teratoma formation, resulting in a possible lethal outcome. Primary ATII cells have shown promise in their ability to reverse lung injury in rats subjected to BLM treatment indicating that endogenous lung cells with progenitor cell properties may prove useful in repairing damaged or diseased lung tissue.
The bleomycin (BLM) mouse model of lung fibrosis is long established and well accepted. Bleomycin is a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus. Bleomycin refers to a family of structurally related compounds that are used as an anti-cancer agent, the chemotherapeutical forms are primarily bleomycin A2 and B2. The drug is used in the treatment of Hodgkin lymphoma (as a component of the ABVD regimen), squamous cell carcinomas, and testicular cancer, as well as in the treatment of pleurodesis and plantar warts. A serious complication of bleomycin exposure, in humans or other mammals, is pulmonary fibrosis and impaired lung function. The anti-neoplastic drug BLM, when administered intra-tracheally to mice, primarily targets the pulmonary epithelium and reproduces the pattern and numerous features of acute lung injury in humans, including rapid onset of inflammation, alveolar injury with fibrosis, and severe hypoxemia.
It is extremely difficult to obtain sufficient quantities of human ATII cells that could be used to clinically treat lung disease. Therefore, to provide an adequate source of transplantable human ATII cells the inventors recently developed a reliable culture and genetic selection procedure to generate a pure population of transplantable ATII cells from hESCs (as described in co-pending U.S. patent application Ser. No. 12/527,969 filed Aug. 20, 2009, the disclosure of which is hereby incorporated herein by reference.)
The present disclosure describes some embodiments in which the use of these hES-ATII cells in an animal model of bleomycin (BLM)-induced acute lung injury for their ability to functionally repair damaged lung tissue without causing teratoma formation. In this model, a relatively high dose of BLM (3.5 units/kg) was given to SCID mice by intra-tracheal intubation. This dose of BLM was used so that at the time of hES-ATII cell transplantation the lung damage resembled that of acute lung injury, including extensive interstitial inflammatory cell infiltration, interstitial thickening, and collagen deposition. Moreover, 3.5 units/kg of BLM caused sufficient lung injury so that endogenous cell repair was negligible and ineffective, leading to 100% mortality of all BLM-treated mice by 13 days post-BLM challenge, thereby allowing the impact of transplanted hES-ATII cells on survival to be evaluated without possible confounding results due to endogenous lung stem cell repair.
Failure to efficiently repopulate the alveolar epithelium after injury is a major factor in the development of pulmonary fibrosis. Alveolar cell damage results in denuded epithelial basement membranes and the release of chemoattractant molecules, which mediate the migration of fibroblasts into the alveolar space and the development of intra-alveolar fibrosis.
During the present studies considerable evidence was obtained to suggest that the transplanted hES-ATII cells were physically engrafted into the mouse alveolar epithelial basement membranes denuded by BLM injury. For example, numerous hES-ATII cells remained in the exhaustively lavaged mouse lung sections obtained 10 days following acute injury, many of the transplanted hES-ATII cells had differentiated into cells expressing phenotypic markers of ATI cells, and no hES-ATII cells were found when transplanted into mouse lungs not subjected to BLM-induced acute injury, i.e., lack of denuded basement membranes to facilitate engraftment. Therefore, re-epithelialization of BLM injured alveolar epithelium by engrafted hES-ATII cells likely prevented the development of severe fibrosis observed in the BLM-treated mice not provided hES-ATII cells. While not wishing to be limited to any single theory to explain the mechanism by which these results are obtained, it is proposed that engraftment and differentiation of hES-ATII cells played a major role in repairing or preventing tissue damage caused by BLM.
Engraftment and differentiation of these cells alone may not have accounted for all the noted therapeutic benefits provided by the hES-ATII cells. It is possible that engraftment is one part of a complex series of biological events initiated by the transplantation of hES-ATII cells. For example, bone marrow stem cells have been reported to cause the release or production of anti-inflammatory cytokines, thereby causing some reversal of lung injury despite their lack of engraftment Also perhaps transplanted hES-ATII cells secrete factors that are either anti-inflammatory or cause the release of anti-inflammatory molecules as well, thereby arresting the inflammation mediated tissue damage and fibrosis caused by BLM.
Although endogenous progenitor or stem cells of the lung were not sufficient to repair or arrest tissue damage in our model of acute lung injury, perhaps hES-ATII cells through the release of growth factors trigger the expansion of endogenous lung progenitor or stem cells that in concert with the hES-ATII cells re-epithelialize the damaged alveolar epithelium.
Whether or not engraftment and differentiation of the transplanted hES-ATII cells accounted for most of the lung repair, it was unequivocal that their presence in the damaged alveolus provided a major therapeutic benefit to animals subjected to BLM-acute lung injury. For example, the loss of body weight by BLM-treated mice was irreversible resulting in death if the mice were not provided hES-ATII cells. However, when given hES-ATII cells the BLM-injured mice experienced an almost complete reversal of weight loss, indicating that the transplanted hES-ATII cells were required for body weight recovery. In addition, treatment with hES-ATII cells not only prevented or reversed visual hallmarks of pulmonary injury, but also restored near normal lung function to mice subjected to BLM acute lung injury. Importantly, the therapeutic benefits provided by hES-ATII cells were long term and without teratoma formation, indicating that the cultures of hES-ATII cells used in these studies were highly pure and free of any remaining hESCs.
The present disclosure provides compositions and methods that were used to obtain evidence for the first time that lung progenitor ATII cells derived from hESCs can be transplanted into acutely damaged alveoli and that these hES-ATII cells arrested or reversed BLM-induced pathogenesis of acute lung injury, including fibrosis. Moreover, the therapeutic benefit provided by the transplantation of hES-ATII cells was long term and with no teratoma side effects.
TransgenesA transgene is an artificial gene that incorporates all appropriate elements critical for gene expression. In general, each transgene contains a promoter, an intron (such as, but not limited to, a rabbit β-globin intron or SV40 intron), a protein coding sequence (termed the reporter, often a selectable marker exon, encoding drug resistance, for example βgeo, neomycin (neo) or puromycin (puro), and transcriptional stop sequence (such as, but not limited to, that from SV40 or human growth hormone, or combinations of sequences). These elements are typically assembled in a bacterial plasmid, and sequences are usually chosen from previous transgenes with proven function. In addition, the construct must be linearized and prokaryotic sequences removed before injection into a stem cell nucleus, such as ES cells or iPS cells. The described promoter was derived from the SPC sequence. This transgene promoter is a regulatory sequence that determines that the transgene will be active only in ATII cells where the SPC gene promoter is activated.
Also encompassed are the general technologies of targeting mutations into the genome of cells, and the process of generating trangenic cell lines from genetically altered ES or iPS cells with specific genetic lesions are well known. A random method of generating genetic lesions in cells (called gene, or promoter, trapping) has been developed in parallel with the targeted methods of genetic mutation such as gene trapping provides a means to create a collection of random mutations by inserting fragments of DNA into transcribed genes.
Although, the use of specific selectable markers have been disclosed and discussed herein, the present invention is in no way limited to the specifically disclosed markers. Additional markers (and associated antibiotics) that are suitable for either positive or negative selection of eukaryotic cells are well known to the art such as those disclosed, inter alia, in Sambrook et al. (1989) Molecular Cloning Vols. I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1989) John Wiley & Sons, all Vols. and periodic updates thereof, as well as Table I of U.S. Pat. No. 5,464,764. Any of the disclosed markers, as well as others known in the art, may be used to practice the present methods.
In addition to the selection systems exemplified herein, a number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes, which can be employed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin, among others.
Although specific transgene vectors have been exemplified, embodiments of the method are by no means to be limited to such vectors. Several different types of vectors that may also be used to incorporate relatively small engineered exons into a target cell transcripts include, but are not limited to, vectors based on recombinant adenovirus, recombinant adeno-associated virus, recombinant SV40, recombinant Moloney murine leukaemia virus, recombinant lentivirus, recombinant herpes simplex virus, recombinant vaccinia virus, and papilloma virus vectors. DNA vectors may be directly transferred into the target cells using any of a variety of chemical or physical means such as, but are not limited to, lipofection, chemical transfection, electroporation, DNA-ligand conjugates, liposomes and virosomes, direct DNA injection, and ballistic (gene gun) delivery.
When it is desired that the transgene be integrated into the chromosomal site of the endogenous copy of the gene, gene targeting may be preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene (i.e., “knockout” animals). In this way, the expression of the endogenous gene may also be eliminated by inserting non-functional sequences into the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type can be done. The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest.
In addition, particularly useful vectors that can be used to introduce the SPC promoter-neo fragment into stem cells, such as ES or iPS cells, and there facilitate the selection of stem cell derived ATII cells include but are not limited to the following commercial vectors available from CLONTECH, all of which encode pGK-puro resistance gene: pMSCVpuro vector (Clontech, Cat#634401); pRetroQ-AcGFO1 N1 VECTOR (Clontech, Cat#632505); and pRetroQ-AcGFO1 Cl VECTOR (Clontech, Cat#632506).
Methods of Repairing Injured or Diseased Alveolar Epithelial TissueAn in vivo method of repairing injured or diseased alveolar epithelial tissue in the lung of a mammal includes transplanting into the lung, at a site comprising injured or diseased alveolar epithelial tissue, a population of differentiated embryonic stem cells, or progeny thereof, at least 99% of which have ATII phenotype, wherein the population of cells is prepared in accordance with an above-described method, and is effective to repair at least a portion of the injured or diseased alveolar epithelial tissue at the site. The differentiated embryonic stem cell, or progeny thereof, may comprise a transgene, which encodes a desirable gene product (e.g., a therapeutic protein or peptide), operably linked to a cell-specific promoter.
Methods of Treating a Genetic Disease Affecting Alveolar epithelial Tissue
An in vivo method of treating a genetic disease affecting alveolar epithelial tissue in the lung of a mammal comprises transplanting into the lung, at a site comprising alveolar epithelial tissue detrimentally affected by the genetic disease, a population of differentiated embryonic stem cells, or progeny thereof, at least 99% of which have ATII phenotype. This population of cells is prepared as described above. The differentiated embryonic stem cell, or progeny thereof, comprises a transgene that encodes a gene product which ameliorates the genetic disease or its detrimental effects in the alveolar epithelial tissue at least at the site of implantation when expressed in vivo. An embryonic stem cell, or its progeny may comprise a transgene operably linked to a cell-specific promoter, wherein the transgene encodes a therapeutic gene product.
EXAMPLESThe following section provides further details regarding examples of various embodiments. Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present invention to its fullest extent. Those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Thus, for example, although the described embodiments illustrate use of human stem cells, those of skill in the art would readily recognize that these methods and compositions could also be applied to veterinary medicine and be used with stem cells from other mammals. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the preferred embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention.
Example 1 Derivation of Alveolar Epithelial Type II (ATI!) Cells from Stem Cell LinesThe disclosed compositions and methods were used to reliably generate a culture of human ES cell-derived ATII cells using a culture and genetic selection procedure that reliably yielded an essentially pure population of human ATII cells. These hES cells were differentiated and cultured in the presence of neomycin to produce sufficiently pure preparations of hES-derived ATII cells with biological and phenotypic characteristics of ATII cells, which included expression of surfactant proteins A, B, and C, presence of lamellar bodies, and the ability to proliferate and differentiate into ATI cells. These hES cell-derived ATII cells are sufficient in numbers and purity such that they could be used to treat and repair damaged lung tissue upon transplantation without the fear of teratoma formation, due to the presence of residual non ATII hES cells. The process by which these exemplary hES-ATII cells were generated is described below.
The structure of the human SPC promoter-neomycin transgene (3′-hprt-SPCP.NEO) is depicted in Figure. 1A. The hES cell line, H9.2, was transfected with the linearized transgene prepared as follows. A 3.8 kb fragment of human genomic DNA containing the human SPC promoter and 170 by of non-coding sequence of exon 1 (Gou et al., Nucleic Acids Res 32, e134, 2004) was cloned into the Asc I site of the 3′-hprt insertion targeting vector (Zheng, B., Mills, A. A. & Bradley, A. Nucleic Acids Res 27, 2354-60, 1999: a gift from Dr. Allan Bradley, The Wellcome Trust Sanger Institute, UK). The Ned cDNA-poly A fragment was sub-cloned into an engineered Nde I restriction site downstream of the SPC promoter. The resulting vector (3′-hprt-SPCP.NEO) is depicted in
The NIH approved human embryonic stem (hES) cell line, H9.2 (passages 45-65) (WiCell, Madison, Wis.), was used throughout. Undifferentiated hES cells were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in 6-well plates with hES cell culture medium, containing 80% Dulbecco's modified Eagle's medium (DMEM)/F12, 20% knockout serum replacement (Gibco Invitrigen), 1% non-essential amino acid, 1mM L-glutamine (CHEMICON), 0.1 mM 2-mercaptoethanol, and 4ng/ml basic fibroblast growth factor (GIBCO INVITROGEN). The hES cells from one 6-well plate were re-suspended in 100 μl of supplemented Mouse ES Cell Nucleofector Solution and mixed with 5 ug of the linearized 3′hprt-SPCP.NEO vector and transfected using the cell Nucleofector™ II (AMAXA). The hES cells were then plated on Matrigel® coated 10-cm plates with MEFs conditioned hES cell medium (MEF-CM) (Xu et al., Nat Biotechnol 19, 971-4, 2001). The 3′-hprt-SPCP.NEO transfected hES cells were selected in the presence of 0.25 μg/ml puromycin (SIGMA) for 14 days. Surviving ES clones were examined for the Neor gene by PCR analysis, and a positive clone (SPCP/Neo.74) containing a single copy of the transgene was selected for further investigation.
To induce spontaneous differentiation in vitro, H9.2 and SPCP/NEO.74 cell lines were cultured in 6-well extra low attachment plates for 5 days to form EBs. To induce spontaneous differentiation via EB formation, collagenase IV dissociated hES cells were plated on 6-well ultra low attachment plates in hES cell medium. On day 2, the resultant EBs were collected, washed, and cultured on fresh 6-well ultra low attachment plates with Differentiation Medium (DM), composed of 80% knockout DMEM (GIBCO INVITRIGEN), 20% FBS, 1% non-essential amino acid, 1mM L-glutamine, penicillin (100 u/ml), and streptomycin (100 μg/ml). On day 6, the EBs were collected and seeded on gelatin-coated 6-well culture plates in DM (15 EBs per well) and allowed to expand. Selection of hES cell derived ATII cells was started on day 6 by adding 20 μg/ml G418 (GIBCO). To promote the differentiation without EB formation, the collagenase IV dissociated hES cells were seeded on Matrigel® coated 6-well plates with MEF-CM (day 0). On day 1, the medium was replaced by DM with or without G418 (20 μg/ml).
Example 2 Confirmation of Stem Cell Derived-Alveolar Type II (ATII) Cell IdentityTo clearly demonstrate that the hES derived ATII cells bore the characteristics of ATII cells several methods were employed to demonstrate features consistent with the phenotype of ATII cells.
PCR: The presence of ATII cells in the differentiating cultures of both hES cell lines was determined by RT-PCR specific for SPC RNA. Total RNA was isolated from the hES cultures using RNA Bee™ (TEL-TEST, Inc.) following the manufacturer's protocol. Appropriate forward and reverse primers and probes were made for each target sequence based on Accession numbers: Total RNA was isolated from the hES cultures using RNA Bee™ (TEL-TEST, Inc.) following the manufacturer's protocol.
Primer pairs were used in the RT-PCR reactions, employing 0.5 μg total RNA and the OneStep RT-PCR kit (QIAGEN). Appropriate forward and reverse primers and probes were made for each target sequence based on Accession numbers: NM—003018 for human surfactant protein C (SPC), mRNA; NM—000492 for human cystic fibrosis transmembrane conductance regulator (CFTR), mRNA; NM—000295 for human peptidase inhibitor (alpha-1 antiproteinase, antitrypsin (alpha-1 -AT)), mRNA; and NR—003286 for human RNA, 18S (18S) ribosomal RNA.
No SPC RNA was detected in the undifferentiated hES cells (day 0) or in the differentiating cultures on day 10. SPC RNA was detected in differentiating cultures of H9.2 and SPCP/NEO.74 hES cells by day 15, with significant increases of SPC RNA observed in both hES cultures on day 25 (
The differentiated hES cell cultures without EB formation (days 10 and 15) were also examined for alveolar epithelial type II specific SPC protein expression using flow cytometry. Differentiated hES cells were dissociated into single cell suspensions by incubation with 0.25% trypsin for 2 min. The dissociated cells were resuspended (0.3×106 cells) in 250 μl Fixation/Permeabilization solution (Cytofix/Cytoperm kit: BD BIOSCIences), kept on ice for 20 min, and washed twice with Perm/Wash™ buffer. After blocking with 10% goat serum in 300 μl Perm/Wash™ buffer for 45 min on ice, the cells were incubated with rabbit anti-human proSPC antibody (1:200 dilution, CHEMICON) in the block solution for 45 min on ice. The cells were resuspended in 350 μl of Perm/Wash™ buffer after incubated with goat anti-rabbit IgG conjugated by R-Phycoerythrin (1:300 dilution, Sigma) for 45 min on ice and washed twice, and analyzed by flow cytometry. For immunofluorescent staining, the differentiated hES cells, with or without G418 selection, were dissociated and seeded on poly-D-lysine coated cover slips, cultured for 24 hr and stained with the rabbit anti-human proSPC antibody following the manufacturer's directions. The SPC positive cells were visualized with Alexa Fluor 546 conjugated goat anti-rabbit IgG (1:1000: MOLECULAR PROBES) with DAPI counterstaining The number of SPC positive cells was counted per 500 cells based on the DAPI staining on each slide. This procedure was also used for immunostaining of surfactant protein A (SPA) and surfactant protein B (SPB) using rabbit anti-human SPA and anti-human proSPB (1:1000: CHEMICON).
These studies revealed low level SPC protein expression in the differentiated culture of H9.2 cells on day 10, which did not increase on prolonged culture (15 days) (as shown in
The synthesis of SPC is a unique feature of ATII cells and is commonly used to identify and differentiate these cells from other lung parenchymal cells. Undifferentiated hES cells, H9.2 and SPCP/NEO.74 bearing hES cells, did not stain positive for SPC (
Percentage of hES-ATII Cells in Differentiated Cell Cultures:
SPC staining was used to determine the percentage of ATII cells in differentiated hES cell cultures. SPC positive staining was displayed in only 11.2% of the differentiated hES cells in the non-selected H9.2 ES cell culture (Table 1). Similar results were observed in the non-selected SPCP/NEO.74 cell culture (12.6%). In stark contrast, 99.6% of cells in the G418 selected differentiated culture of SPCP/NEO.74 cells expressed SPC protein, indicating that G418 selection of the transfected hES cells produced an essentially pure culture of ATII cells.
Identification of Lamellar Bodies in hES-ATII Cells:
Lamellar bodies are unusual intracellular organelles that contain pulmonary surfactant proteins and lipids. The presence of lamellar bodies is a criterion traditionally used for the identification of ATII cells. To confirm that hES-ATII cells contained these intracellular organelles, G418 selected hES-ATII cells were examined by transmission electron microscopy.
The G418 selected hES cell derived ATII cells and A549 cells were trypsinized and fixed (2 hr) in suspension with 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and then post-fixed in 1% tannic acid (5 min) followed by 1% osmium tetroxide (1 hr) and then aqueous uranyl acetate (1 hr). Samples were subsequently dehydrated in a graded ethanol series, embedded in Araldite resin and ultrathin serial sections (100 nm) were obtained using an ultramicrotome (RMC 7000, RMC, AZ) equipped with a diamond knife. Sections were stained with uranyl acetate and lead citrate before photographing with a JEOL 200CX electron microscope. The hES-ATII cells exhibited ultrastructural features characteristic of human alveolar type II cells, including cytoplasmic organelles with clear appearance of Lamellar bodies (
Because ATII cells express Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), Alpha-1-Antitrypsin (α-1AT) in vivo and play an important role in alveolar homeostasis, hES-ATII cells were examined for expression of CFTR and α-1AT RNA by RT-PCR. As anticipated, specific RNA transcripts of CFTR and α-1AT were observed in the hES-ATII cells and A549 (ATII) cells, but not in the starting undifferentiated hES cell lines (
Cultures (4, 9, 11, or 14 days) of differentiating hES cell-derived ATII cells and A549 cells were switched to DMEM with 15% FBS, incubated for 24 hrs, and 100 μl samples of each culture added to 96-well plates, which had been coated with either anti-human C3c or anti-human C5 antibodies (2 μg/ml Quidel). After incubation at room temperature for 2 hrs, the plates were exhaustively washed, incubated for 2 hrs with the primary goat anti-human C3 and anti-human C5 antibodies (Complement Technology, Inc.), washed, and incubated for 1 hr. with alkaline phosphatase conjugated rabbit anti-goat IgG (Sigma). Samples were developed using the Alkaline Phosphatase Yellow Liquid Substrate System (Sigma).
ELISA based measurements of the cell culture supernatants indicated that early differentiated ATII cells (day 10) synthesized and secreted C3 at a rate of 33±3 ng/mg/24 hr, which was comparable to that produced by the human alveolar type II cell line A549 (data not shown). Similar levels of C3 were also observed on day 12 and day 15 (FIG. 6,C). C5 was also detected but only in the day 15 cultures (1.6 ng/mg/24 hr) (FIG. 6,D). The amount of C5 produced by the hES-ATII cells was similar to the quantity of C5 reported by Strunk et al., 1988 (J Clin Invest 81, 1419-26, 1988) to be produced by primary cultures of rat and human ATII cells.
Example 3 Theraputic use of hES-ATII CellsThe hES derived-ATII cells were produced in sufficient in numbers and purity such that they could be used to treat and repair damaged lung tissue upon transplantation. Transplantation the hES derived-ATII cells prepared by the disclosed compositions and methods after lung injury greatly reduced the extent of damage within the lung, as evidenced by the presence of only a few isolated, small areas of injured tissue surrounded by much larger areas of normal alveolar structure. This therapy also improved lung function, as evidenced by the reversal of the body weight loss that accompanies acute lung injury and a reversal of injury impaired lung tidal volumes and blood arterial oxygen saturation levels to normal levels.
Stable Transfected Human Stem Cell Lines and Production of Pure hES-ATII Cells.
Aquaporin 5 (AQP5) is a mercury-sensitive water channel that is found in the apical membrane of ATI cells with little to no expression in ATII cells. T1α is a cell surface mucin-like glycoprotein with no known function that is expressed by ATI cells but not ATII cells. Both AQP5 and T1α are used frequently as phenotypic markers for ATI cells. Therefore, to visualize the differentiation of hES-ATII cells into ATI cells two new stable hES cell lines (AQP5P.65 and T1αP.53) were generated in which either the AQP5 or T1α genomic promoters were cloned upstream of the LacZ gene (
To construct a human SPC promotor-NEOr+T1α/AQP5 promoter-LacZ transgene vectors. The 3′hprt.SPCP.NEOr vector containing the human SPC promoter-NEOr transgene and puromycin resistant gene (
The NIH approved human embryonic stem (hES) cell line, H9.2 (passages 40-65) (WiCell, Madison, Wis.), was used throughout this study. Undifferentiated hES cells cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in 6-well plates were transfected with either the 3′hprt.SPCP.NEOr.T1αP.LacZ or the 3′hprt.SPCP.NEO.AQP5P.LacZ vectors using the Nucleofector™ II (AMAXA, Gaithersburg, Md.), and selected in the presence of 0.25 μg/ml puromycin (SIGMA, St. Louis, Mo.) for 14 days as described previously.
Surviving hES clones were examined for the Neor and LacZ transgene by PCR analysis. The hES clones T1α.LacZ.53 and AQP5P.LacZ.65 containing only a single copy of the corresponding transgene were selected for further analysis.
In vitro Differentiation and Selection of hES-ATII Cells.
The hES cell lines, SPCP.NEO.74, T1α.LacZ.53, and AQP5P.LacZ.65, were cultured on Matrigel® (BD BIOSCIENCES) coated 10-cm plates and allowed to spontaneously differentiate in differentiation medium (DM) composed of 80% knock-out DMEM (GIBCO INVITROGEN), 20% FBS, 1% nonessential amino acids, 1 mM L-glutamine, 100 ug/ml penicillin, and 100 ug/ml streptomycin. The hES-ATII cells in the cultures were selected with G418 (20 μg/ml, GIBCO) as described in Wang et al., 2007 (A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 104, 4449-4454, 2007). For transplantation, G418 selected hES-ATII cells on day 14 were trypsinized and then seeded back onto fresh matrix gel coated 10 cm culture plates with DM. The hES-ATII cells were harvested the following day, washed once with normal saline, and re-suspended in normal saline (107 cells/ml). For spontaneous differentiation studies, G418 selected hES-ATII cells as well as normal human ATII cells were seeded onto 6-well culture plates with DMEM (GIBCO INVITROGEN) containing 10% FBS (day 0) with medium change every other day. The primary ATII cells were isolated and cultured as described in (Alcorn et al., Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses. Am J Respir Cell Mol Biol 17, 672-682, 1997). Total RNA was isolated on days 0, 2, 4, 6, and 8 to analyze the expression of human ATI phenotypic markers, T1α and AQP5, by QRT-PCR. To examine the ability of hES-ATII cells to proliferate in vitro, G418 selected hES-ATII cells were re-suspended in mouse embryonic fibroblast (MEF) conditioned DMEM medium containing 10% FBS with or without 30 ng/ml of rhKGF (R&D Systems) and plated on Matrigel® coated 6-well plates (1.0×104/well). The medium was changed every day for 8 days. Matrigel® (BD BIOSCIENCES) is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. This mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. The chief components of Matrigel® are structural proteins such as laminin and collagen which present cultured cells with the adhesive peptide sequences that they would encounter in their natural environment. Also present are growth factors that promote differentiation and proliferation of many cell types. Matrigel® Basement Membrane Matrix is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins to include laminin (a major component), collagen IV, heparan sulfate proteoglycans, and entactin/nidogen. BD Matrigel® Matrix also contains TGF-beta, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor. BD Matrigel® Basement Membrane Matrix promotes attachment and differentiation of both normal and transformed anchorage dependent epithelioid and other cell types.
Proliferation and Differentiation of hES-ATII Cells in vitro.
One of the important biological functions of ATII cells is to proliferate and to serve as progenitor cells for ATI cells. To determine if hES-ATII cells proliferated in vitro, hES-ATII cells generated from the SPC/NEO.74 hES cell line were cultured with MEF conditioned DMEM medium. Recombinant Keratinocyte Growth Factor (rhKGF), also referred to as FGF in
To assess the ability of hES-ATII cells to differentiate into ATI cells in vitro, hES-ATII cells and control primary human ATII cells were cultured with DMEM and left to spontaneously differentiate. Differentiation was assessed by real-time QRT-PCR using specific primer pairs to detect gene expression of AQP5 and T1α. Total RNA was isolated from the differentiating cultures of hES-ATII cells and primary human ATII cells on days 0, 2, 4, 6, and 8 using RNA Bee (TEL-TEST, Friendswood, Tex.). TaqMan One-Step RT-PCR Master Mix Kit control 18S rRNA was used. Appropriate forward and reverse primers and probes were made for each target sequence based on NCBI Accession numbers: NR—003286 for human RNA, 18S ribosomal 1 (AB APPLIED BIOSYSTEMS) was used for QRT-PCR analysis following manufacturer's instructions employing 100 ng of total RNA for AQP5 and T1α and 100 pg of total RNA for endogenous 18S, ribosomal RNA. Appropriate forward and reverse primers and probes were made for each target sequence based on Accession numbers: BC032946 for human aquaporin 5 (AQP5), mRNA; NM—001006625 for human sapiens podoplanin (PDPN), transcript variant 4, mRNA (T1 alpha) and NR—003286 for human RNA, 18S (18S) ribosomal RNA. For example, the following primers and probes were employed: (1) AQP5 forward (5′-CCA TGG TGG TGG AGC TGA TTC TG-3′) (SEQ ID NO: 9), AQP5 reverse (5′-TG CGG CGG GAG TCA GT-3′) (SEQ ID NO: 10) and AQP5 probe (5′-6-FAM-CTT CCA GCT GGC ACT CTG CAT CTT CGC C-TAMRA-3′) (SEQ ID NO: 11); (2) T1α forward (5′-GCT GCT TTG TTC TGG AAT ATG GAT ATC TC-3′) (SEQ ID NO: 12), T1α reverse (5′-TTG AGC CTC TAG CAC CAT TAA GCA-3′) (SEQ ID NO: 13) and T1α probe (5′-FAM-AGC AGC TTC CTC GGC ATC CAG G-TAMRA-3′) (SEQ ID NO: 14); and (3) 18S forward (5′-TAA CGA ACG AGA CTCTGG CAT-3′)(SEQ ID NO: 15), 18S reverse (5′-CGG ACA TCT AAG GGC ATC ACA G-3′) (SEQ ID NO: 16) and 18S probe (5′-FAM-TGG CTG AAC GCC ACT TGT CCC TCT AA-TAMRA-3′) (SEQ ID NO: 17).-QRT-PCR was carried out at 48° C. for 30 min and 95° C. for 10 min followed by 40 cycles at 95° C. for 15 sec and 60° C. for 1 min in a 7900HT Sequence Detection Systems (APPLIED BIOSYSTEMS).
As shown in
The differentiating cultures of hES-ATII cells on days 0, 2, 4, 6, and 8 were washed 3 times with PBS before fixation in 0.5% glutaraldehyde in PBS for 10 min at room temperature. After washing with PBS containing 1 mM MgCl2, the cells were incubated overnight at 37° C. with X-gal solution composed of 0.1% X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mM MgCl2 in PBS. β-galactosidase activity was visualized with X-gal precipitates after washing with PBS. For LacZ staining of lung tissue (Kotton, D. N. et al. Bone marrow-derived cells as progenitors of lung alveolar epithelium. Development 128, 5181-5188, 2001), lungs which had been lavaged 3 times with 500 μL of PBS containing 2mM MgCl2, were fixed for 10 min by instillation of 0.5% glutaraldehyde in PBS via a tracheal catheter. The fixative was removed by washing 3 times with PBS containing 2 mM MgCl2 through the tracheal catheter. Each lung was instilled with 500 μl of X-gal solution before tracheal ligation and then immersed in X-gal solution overnight at 37° C. Lungs were fixed again in 4% paraformaldehyde at 4° C. overnight, embedded in paraffin and sectioned. The lung sections were counterstained with Nuclear Fast Red (Vector Laboratories, Inc) before mounting with Cytoseal (Richard Allan Scientific).
Consistent with the evidence obtained by QT-PCR and shown in
Treatment of Injured Lungs with Human Stem Cell Derived-ATII Cells.
The bleomycin (BLM) mouse model of lung fibrosis is long established and well accepted. BLM is a glycopeptide antibiotic produced by the bacterium Streptomyces verticillus. BLM refers to a family of structurally related compounds that are used as an anti-cancer agent, the chemotherapeutical forms are primarily bleomycin A2 and B2. The drug is used in the treatment of Hodgkin lymphoma (as a component of the ABVD regimen), squamous cell carcinomas, and testicular cancer, as well as in the treatment of pleurodesis and plantar warts. A serious complication of bleomycin exposure, in humans or other mammals, is pulmonary fibrosis and impaired lung function. To impair possible graft rejection of the hES-ATII cells, the BLM-induced acute lung injury model was established using immune deficient SCID mice. Pathogen free, 8 to 10 week old, female SCID mice with body weights of 16-18 g, on a C57BL/6 genetic background (Jackson Laboratories, Bar Harbor, Me.) received 50 μl of either BLM (3.5 units/kg, Bristol-Myers Squibb Company) or sterile normal saline endotracheally via oropharynx intubation (Brown, R. H., Walters, D. M., Greenberg, R. S. & Mitzner, W. A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice. J Appl Physiol 87, 2362-2365, 1999) using a BioLITE Intubation Illumination System (Braintree Scientific Inc). Oropharynx intubation required no surgery and thus no tracheal inflammation, thereby allowing subsequent noninvasive administration of hES-ATII cells on days 1 or 2 after BLM challenge. The BLM treated mice were transplanted with hES-ATII cells derived from the three different stable transfected hES cell lines, SPCP.NEO.74, T1α.LacZ.53, and AQP5P.LacZ.65, at a dose of 0.5×106 cells in 50 μl of sterile normal saline via an endotracheal catheter as above. Control mice received 50 μl of sterile normal saline. The body weights were determined every another day. BLM exposed mice with or without transplantation of hES-ATII cells were sacrificed on day 10 after euthanized and lungs harvested for histological analysis.
Histological Analysis of Lung Tissue.On day 10 after BLM challenge, the mouse lungs were lavaged 3 times with 500 μL of PBS, inflated and fixed with 500 μl of 4% paraformaldehyde, and then immersed in the fixative solution at 4° C. overnight. After embedding in paraffin, tissue sections from three different levels of each lung were prepared on slides. Collagen deposition was evaluated using Sirius Red staining as described by Chu et al., 1998 (Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 158, 1936-1944, 1998). Briefly, lung sections were incubated with 1% Sirius Red and 0.1% Fast Green FCF (Sigma Aldrich) in saturated picric acid for one hr. The slides were then washed twice with 0.5% acetic acid in distilled H2O. The slides were then dehydrated and mounted with Cytoseal. Another set of slides from the same lung were stained with hematoxylin-eosin for routine histology. BLM induced lung damage was graded according to the lung area (0, <10, 10 to 25, 25 to 50, 50 to 75 and >75%) involved with cellular infiltration, interstitial thickening, structure distortion as well as abnormal collagen deposition (Chen et al., Attenuation of lung inflammation and fibrosis in interferon-gamma-deficient mice after intratracheal bleomycin. Am J Respir Cell Mol Biol 24, 545-555, 2001).
BLM when administered intra-tracheally to mice primarily targets the pulmonary epithelium and reproduces the pattern and numerous features of acute lung injury in humans, including rapid onset of inflammation, alveolar injury that heals with fibrosis, and severe hypoxemia. As described, female SCID/C57BL/6 mice were subjected to BLM-induced acute lung injury and following BLM treatment, hES-ATII cells were administered by intra-tracheal intubation into the mouse terminal airways, and end point lung sections were examined by immunohistochemistry.
Lung section slides were prepared as described above were deparaffinized, hydrated, and incubated with 20 ug/ml proteinase K solution containing 50 mM Tris and 1 mM EDTA, pH 8.0 at 37° C. for 15 min. After rinsing twice in PBST (0.05% Tween-20 in PBS), slides were incubated in 1% Triton X-100 in PBS for 30 min before blocking for one hr. with 5% of normal goat serum in PBS containing 0.2% Triton X-100. To block the endogenous mouse IgG, the sections were further incubated with 0.12 mg/ml of unconjugated AFFINIPURE Fab fragment goat anti-mouse IgG (H+L) (Jackson Immunoresearch Labs, code#:115-007-003) for one hr. To identify hES-ATII cells, the sections were incubated with 1:10 diluted mouse anti-human nuclei monoclonal antibody and 1:100 diluted rabbit anti-human pro-SPC antibody (CHEMICON) in PBS for one hr. The human nuclei and SPC positive cells were visualized with Alexa Fluor 546 F(ab′)2 fragment of goat anti-mouse IgG (H+L) and Alexa Fluor 488 F(ab′)2 fragment of goat anti rabbit IgG (H+L), respectively. To determine if any of the transplanted hES-ATII cells had differentiated into cells expressing the ATI phenotypic marker T1α, immunofluorescent staining was performed with the mouse anti-human nuclei monoclonal antibody and a 1:50 diluted rabbit anti-human T1α antibody (ABGENT), and secondary antibodies as above.
Transplanted hES-ATII cells were identified using an anti-human pro-SPC antibody and a mouse anti-human nuclei monoclonal antibody (Chen et al., Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats. Stroke 32, 2682-2688, 2001; Vescovi et al., Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 156, 71-83, 1999). As shown in
Treatment with Human Stem Cell Derived-ATII Cells Abrogates Acute Lung Injury.
Following intra-tracheal exposure to BLM, hES-ATII cells were transplanted into the mouse injured alveoli to determine if the hES-ATII cells would prevent or reverse the acute lung damage caused by BLM. Direct initial damage to the alveolar epithelial cells occurred followed by acute inflammation within 24 hrs. Ten days following BLM challenge, approximately 50 to about 70% of the lung alveolar epithelium was severely injured, noted by interstitial thickening, alveolar collapse, cystic air spaces, extensive interstitial infiltration of inflammatory cells and collagen deposition (
BLM-induced lung injury is associated with significant loss of body weight. To determine if the transplantation of hES-ATII cells would either arrest or reverse this loss of weight in BLM challenged mice, mice with or without hES-ATII cell therapy were weighed for 10 days following BLM challenge (
Treatment with Human Stem Cell Derived-ATII Cells Improves Lung Function.
BLM exposure of the lungs of the SCID mice caused extensive alveolar epithelial cell damage, resulting in airway structure distortion, interstitial tissue thickening, and collagen deposition similar to that associated with human lung disease or damage (
The spontaneous lung tidal volume of BLM injured mice with or without transplanted hES-ATII cells was determined using a rodent pulmonary plethysmograph (Buxco Electronics, Inc., Sharon, Conn.). In addition, blood arterial oxygen saturation was recorded using a small rodent oximeter sensor mounted on the thigh of each tested mouse (MouseOX, STARR Life Sciences). Data were collected for a minimum of 10 sec without any error code for the two measured parameters, six measurements (6×10 sec) per mouse during 3 min period.
As illustrated in
To determine if transplantation of the hES-ATII cells would provide a long term benefit and to ensure that the hES-ATII cells would not lead to teratoma formation, 28 female SCID mice were exposed to 3.5 units/kg of BLM as before; 6 of these mice received hES-ATII cells following BLM challenge. Of the 22 BLM exposed mice, 5 (22.7%) died during the first 7 days, and another 5 died between 7 to 10 days after BLM challenge (
Statistical analysis. The data shown in
In some embodiments of the present invention, a method of treating injured or diseased alveolar epithelial tissue in the lung of a mammal comprises transplanting into the lung a population of differentiated embryonic stem cells, or progeny thereof, sufficiently pure and numerous, at least 95%, and in many cases at least 99% of which have alveolar type II phenotype, effective to repair at least a portion of the injured or diseased alveolar epithelial tissue. In some embodiments the population of differentiated embryonic stem cells, or progeny thereof, are introduced in the lung directly at a site of injury or diseased tissue. In some embodiments, the population of cells is introduced into the injured or diseased lung endotracheally via oropharynx intubation. In certain embodiments the population of cells is prepared by a method that includes (a) culturing the at least one embryonic stem cell in vitro in a medium formulated to produce differentiated cells without formation of an embryonic body, wherein at least some of the differentiated cells are of ATII cell phenotype expressing at least one biomarker of ATII cells. The preparation method further includes (b) detecting expression of the biomarker(s) to identify the differentiated cells of ATII phenotype; and (c) isolating the identified cells. The preparation method also includes (d) cloning the isolated cells to produce the population of cells for transplantation. Although in many cases commercially available MATRIGEL is a preferred component of the in vitro culture medium or is used as a support material for the cells in culture, another suitable attachment substrate material or medium containing extracellular matrix components may be employed instead of MATRIGEL in some embodiments of the above-described method. Any such alternative attachment substrate material promotes maintaining pluripotent stem cells in the undifferentiated state during culturing. In preferred embodiments, the embryonic stem cells are cultured without the use of feeder cells. In some embodiments, the embryonic stem cells are human embryonic stem cells.
In some embodiments, an above-described differentiated embryonic stem cell, or progeny thereof, comprises a transgene operably linked to a cell-specific promoter, wherein the transgene encodes a therapeutic gene product.
In some embodiments, the disease comprises a genetic disease affecting alveolar epithelial tissue in the lung of the mammal, and the population of differentiated embryonic stem cells comprises a transgene operably linked to a cell-specific promoter. In certain embodiments, the transgene encodes a gene product that ameliorates the detrimental effects of the genetic disease in the alveolar epithelial tissue of the mammal.
In various embodiments of an above-described method, the at least one biomarker comprises surfactant protein C, cystic fibrosis transmembrane conductance receptor, α-1-antitrypsin, complement protein C3, complement protein C5, or a combination of any of those.
In some embodiments, an above-described transgene comprises a drug resistance gene that, when expressed, is capable of imparting resistance to the drug in the stem cell or progeny thereof. In some embodiments of the disclosed methods, in (c), isolating the differentiated cells having the ATII cell phenotype comprises selecting a purified population of differentiated cells wherein at least 95%, or in many cases at least 99%, of the cells have ATII cell phenotype. In some embodiments of an above-described method, the population of differentiated embryonic stem cells, or progeny thereof, comprises a sufficient number of sufficiently pure ATII cells (e.g., at least 106 cells within 15 days after differentiation, wherein at least 95%, and in many cases at least 99%, of the population have ATII phenotype). In some embodiments, of an above-described method the mammal is suffering from a primary lung injury, a secondary lung injury a disorder or syndrome that leads to lung inflammation and lung injury or a traumatic lung injury.
Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present invention to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the preferred embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.
Claims
1. A construct comprising a DNA sequence encoding human surfactant protein C promoter operably linked to a DNA sequence encoding at least one drug -resistance gene.
2. The construct of claim 1, wherein said drug-resistance gene is a neomycin-resistance gene.
3. A linear expression vector comprising the construct of claim 2 operably linked downstream of a DNA sequence encoding a 3′-hprt vector, wherein said 3′-hprt vector comprises a puromycin-resistance gene.
4. A transgenic stem cell comprising the expression vector of claim 3.
5. A method of preparing a population of in vitro cultured cells of alveolar epithelial type II (ATII) cell lineage derived from at least one transgenic stem cell, comprising:
- (a) culturing transgenic stem cells in vitro in a medium comprising laminin, collagen and growth factors that promote differentiation and proliferation of said stem cells, to produce differentiated cells without formation of an embryonic body, wherein at least some of said differentiated cells are of ATII cell phenotype, at least one said differentiated cell containing an expressible transgene comprising the DNA construct of claim 1;
- (b) selecting said differentiated cells for drug resistance, to isolate the differentiated cells having ATII cell phenotype from those not expressing the drug resistance; and
- (c) cloning the isolated cells to produce a population of cells at least 95% of which having ATII cell phenotype.
6. The method of claim 5 wherein at least 99% of the produced population of cells have an ATII cell phenotype.
7. The method of claim 5 wherein the transgenic stem cells are transgenic embryonic stem cells.
8. The method of claim 5 wherein said isolated cells express at least one biomarker selected from the group consisting of surfactant protein C, cystic fibrosis transmembrane conductance receptor, α-1 -antitrypsin, complement protein C3 and complement protein C5.
9. The method of claim 5 wherein said transgenic stem cells comprise a therapeutic transgene operably linked to a cell-specific promoter.
10. The method of claim 5 wherein in (c), cloning the isolated cells to produce a population of cells having ATII cell phenotype comprises producing a population of more than 106 cells within 15 days of differentiation, wherein at least 99% of said population have ATII phenotype.
11. A method of preparing a population of in vitro cultured cells of alveolar epithelial type II (ATII) cell lineage derived from at least one transgenic induced pluripotent stem cell, comprising:
- (a) culturing transgenic induced pluripotent stem cells in vitro in a medium comprising laminin, collagen and growth factors that promote differentiation and proliferation of said stem cells, to produce differentiated cells without formation of an embryonic body, wherein at least some of said differentiated cells are of ATII cell phenotype, at least one said differentiated cell containing an expressible transgene comprising the DNA construct of claim 1;
- (b) selecting said differentiated cells for drug resistance, to isolate the differentiated cells having ATII cell phenotype from those not expressing said drug-resistance; and
- (c) cloning the isolated cells to produce a population of cells at least 95% of which having ATII cell phenotype.
12. The method of claim 11, wherein said at least one drug resistance gene comprises a puromycin resistance gene and a neomycin resistance gene.
13. An in vivo method of repairing injured or diseased alveolar epithelial tissue in the lung of a mammal, comprising transplanting into said lung containing injured or diseased alveolar epithelial tissue, a population of differentiated stem cells, or progeny thereof, at least 95% of which have ATII phenotype, wherein said population of cells is prepared in accordance with the method of claim 1, and is effective to repair at least a portion of said injured or diseased alveolar epithelial tissue.
14. The method of claim 13 wherein at least 99% of said population of cells have a ATII cell phenotype.
15. The method of claim 13 wherein said stem cells comprise embryonic stem cells.
16. The method of claim 13 wherein said at least one said differentiated stem cell, or progeny thereof, comprises a therapeutic transgene operably linked to a cell-specific promoter, wherein said transgene encodes a therapeutic gene product.
17. The method of claim 16, wherein said mammal suffers from a genetic disease affecting alveolar epithelial tissue in the lung, and said therapeutic transgene encodes a gene product for ameliorating the detrimental effects of said genetic disease in said alveolar epithelial tissue.
18. The method of claim 13, wherein said transplanting comprises administering said population of cells directly to injured or diseased alveolar epithelial tissue in said lung.
19. The method of claim 13, wherein said transplanting comprises administering said population of cells into said lung endotracheally via oropharynx intubation.
20. The method of claim 13 wherein said isolated cells isolated cells express at least one biomarker selected from the group consisting of surfactant protein C, cystic fibrosis transmembrane conductance receptor, α-1-antitrypsin, complement protein C3 and complement protein C5.
Type: Application
Filed: Jan 18, 2011
Publication Date: Aug 18, 2011
Applicant: BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM (Austin, TX)
Inventors: Rick A. WETSEL (Houston, TX), Dachun WANG (Houston, TX)
Application Number: 13/008,637
International Classification: A61K 35/12 (20060101); A61P 11/00 (20060101); C12N 15/85 (20060101); C12N 5/10 (20060101); A61M 31/00 (20060101);
