Epithelial cell differentiation of human mesenchymal stromal cells

Abstract

To provide methods and compositions for the differentiation of mesenchymal stem cells into lung cells and a population of lung cells.SOLUTION: Provided is a method of differentiating a mesenchymal stem cell (MSC) into a lung cell, the method comprising: seeding MSC on a substrate; and exposing the MSC seeded substrate to a growth medium containing at least one of retinoic acid and human epidermal growth factor, thereby differentiating the MSCs into a lung cell that expresses at least one epithelial marker.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] Cross-reference of related applications This application is entitled to priority under 119(e) of U.S. Patent Provisional Application No. 61 / 866,570, filed on 16 August 2013, which is incorporated in its entirety by reference herein.

[0002] Description of research and development funded by the federal government. This invention was made with government assistance under National Institutes of Health patents GM086287, HL111016, and HL098220. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Various groups have described the ability of bone marrow-derived cells to contribute to lung repair and regeneration (Krause et al., 2001, Cell 105: 1-9 (Non-patent Literature 1); Rojas, et al., 2005, Am J Respir Cell Mol Biol 33(2): 145-52 (Non-patent Literature 2); Kotton, et al., 2001, Development 128(24):5181-8 (Non-patent Literature 3); Wong et al., 2009, Cytotherapy 11:676-687 (Non-patent Literature 4)). Interestingly, these reports demonstrate the contribution of bone marrow hematopoietic stem cell components (HSCs) and mesenchymal stromal cell (MSC) fractions to lung epithelium. In many of these studies, the contribution of bone marrow-derived cells to lung epithelium appears to require lung injury (Krause, 2008, Proc Am Thorac Soc 5:323-327 (Non-Patent Literature 5)). Particularly interesting is the fact that subpopulations of human and rodent bone marrow MSC-like cells can express Clara cell-secreted protein (CCSP), a marker associated with Clara cells in the lung (Wong et al., 2009, Cytotherapy 11:676-687 (Non-Patent Literature 4)). These researchers also showed that tail vein administration of mouse CCSP+ bone marrow cells to CCSP knockout mice resulted in the uptake of CCSP+ cells in the host lung after lung injury.

[0004] Mesenchymal stromal cells derived from bone marrow and adipose tissue have also been shown to play an immunomodulatory role (DelaRosa, et al., 2012, Stem Cells and Development 21: 1333-1343 (Non-Patent Literature 6); Rasmusson et al., 2003, Transplantation 76: 1208-1213 (Non-Patent Literature 7)). These include a lack of T cell activation and a reduction in activated lymphocytes when MSCs are delivered in vivo in animal models (Rasmusson et al., 2003, Transplantation 76: 1208-1213 (Non-Patent Literature 7)). Furthermore, MSCs produce paracrine signals that have been shown to play an anti-inflammatory role in the lungs (Lee et al., 2011, Stem Cells 29:913-919 (Non-Patent Literature 8); Ortiz et al., 2003, PNAS 100:8408-8411 (Non-Patent Literature 9)). From a therapeutic standpoint, the evidence supporting the use of MSCs in the potential treatment of lung diseases, either through direct contributions to lung epithelium or through indirect paracrine immunomodulatory mechanisms, is very interesting.

[0005] Previous studies have utilized neonatal rodent cells for the regeneration of rat lungs created by bioengineering (Petersen et al., 2010, Science 329:538-541 (Non-Patent Literature 10)). These experiments demonstrated the feasibility of using decellularized lungs as a means of directing donor cells to anatomically precise locations, and the resulting, albeit temporary, functionality of organs with rearranged cells. A recent report using mouse bone marrow-derived MSCs deployed in decellularized mouse lungs has also been described (Daly et al, 2012, Tissue Eng Part A 18: 1-16 (Non-Patent Literature 11)). This study failed to demonstrate a significant contribution from mouse MSCs seeded to take on the fate of lung epithelium. Another study using primate-derived MSCs and lung scaffolds also showed no conversion of MSCs to lung epithelial fate after placement on decellularized primate lungs (Bonvillain et al., 2012, Tissue Eng Part A 18(23-24):2437-52 (Non-Patent Literature 12)).

[0006] Therefore, there is a need in the art for compositions and methods for the differentiation of mesenchymal stromal cells into epithelial cells. The present invention addresses this unmet need in the art. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Krause et al., 2001, Cell 105: 1-9 [Non-Patent Document 2] Rojas, et al., 2005, Am J Respir Cell Mol Biol 33(2): 145-52 [Non-Patent Document 3] Kotton, et al., 2001, Development 128(24):5181-8 [Non-Patent Document 4] Wong et al., 2009, Cytotherapy 11:676-687 [Non-Patent Document 5] Krause, 2008, Proc Am Thorac Soc 5:323-327 [Non-Patent Document 6] DelaRosa, et al., 2012, Stem Cells and Development 21: 1333-1343 [Non-Patent Document 7] Rasmusson et al., 2003, Transplantation 76: 1208-1213 [Non-Patent Document 8] Lee et al., 2011, Stem Cells 29:913-919 [Non-Patent Document 9] Ortiz et al., 2003, PNAS 100:8408-8411 [Non-Patent Document 10] Petersen et al., 2010, Science 329:538-541 [Non-Patent Document 11] Daly et al, 2012, Tissue Eng Part A 18: 1-16 [Non-Patent Document 12] Bonvillain et al., 2012, Tissue Eng Part A 18(23-24):2437-52 [Overview of the project]

[0008] As described below, the present invention includes methods and compositions for differentiating mesenchymal stem cells, such as bone marrow and adipose tissue mesenchymal stem cells, into lung cells, populations of lung cells, and methods for mitigating or treating lung abnormalities in subjects where there is a need.

[0009] One aspect of the present invention is a method for differentiating mesenchymal stem cells (MSCs) into lung cells, comprising the steps of seeding MSCs on a substrate; and exposing the substrate seeded with MSCs to a growth medium containing at least one of retinoic acid and human epithelial growth factor, thereby differentiating the MSCs into lung cells expressing at least one epithelial marker.

[0010] Another aspect is a method for regulating the differentiation of mesenchymal stem cells (MSCs) into lung cells, comprising culturing MSCs on a substrate, thereby differentiating the MSCs into lung cells.

[0011] Yet another aspect includes a population of lung epithelial cells differentiated from mesenchymal stem cells (MSCs) in which the lung epithelial cells express at least one epithelial marker selected from the group consisting of CCSP, pro-SPC, and cytokeratin-5.

[0012] Yet another aspect includes a population of lung cells produced by a method for differentiating mesenchymal stem cells (MSCs) into lung cells, comprising culturing MSCs on a substrate, thereby differentiating the MSCs into lung cells.

[0013] In various embodiments of the above aspects or any other aspect of the invention described herein, the MSCs are selected from the group consisting of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs). In one embodiment, the lung cells exhibit at least one characteristic of type II alveolar epithelial cells. In another embodiment, at least one characteristic of type II alveolar epithelial cells is the expression of at least one epithelial marker selected from the group consisting of pro-SPC and cytokeratin-5. In yet another embodiment, the lung cells exhibit at least one characteristic of Clara cells. In yet another embodiment, at least one characteristic of Clara cells is the expression of Clara cell secretory protein (CCSP).

[0014] In one embodiment, lung epithelial cells are seeded on a substrate. In another embodiment, the substrate is decellularized lung tissue. In yet another embodiment, the substrate is a coating comprising an extracellular matrix. In yet another embodiment, the extracellular matrix comprises one or more of human ECM, laminin, fibronectin, collagen IV, and collagen I.

[0015] In one embodiment, the MSC is selected from the group consisting of bone marrow-derived MSC (BM-MSC) and adipose tissue-derived MSC (AT-MSC). In another embodiment, the lung epithelial cells are selected from the group consisting of type I alveolar epithelial cells, type II alveolar epithelial cells, and Clara cells.

[0016] In another embodiment, the MSC is bone marrow-derived MSC (BM-MSC), and the MSC differentiates into cells that exhibit at least one characteristic of type II alveolar epithelial cells. In yet another embodiment, at least one characteristic of type II alveolar epithelial cells is at least one expression selected from the group consisting of proSPC and cytokeratin-5.

[0017] In another embodiment, the MSC is adipose tissue-derived MSC (AT-MSC), and the MSC differentiates into cells that exhibit at least one characteristic of Clara cells. In yet another embodiment, at least one characteristic of Clara cells is the expression of Clara cell secretory protein (CCSP).

[0018] In one embodiment, the population of lung cells comprises genetically modified cells. In another embodiment, the cells are genetically modified to express a therapeutic gene. In yet another embodiment, the genetically modified cells are lung epithelial cells that are genetically modified to express a therapeutic gene.

[0019] Another aspect is a method for reducing or treating a lung abnormality in a mammal, comprising the step of administering a therapeutically effective dose to a mammal of a population of lung epithelial cells differentiated from mesenchymal stem cells (MSCs), wherein the lung epithelial cells express at least one epithelial marker selected from the group consisting of CCSP, proSPC, and cytokeratin-5.

[0020] Another aspect includes administering a therapeutically effective dose to a mammal of a population of lung cells produced by a method of differentiating mesenchymal stem cells (MSCs) into lung cells, wherein the differentiation method includes culturing MSCs on a substrate and thereby differentiating the MSCs into lung cells, and is a method of reducing or treating lung abnormalities in mammals. [Invention 1001] A method for differentiating mesenchymal stem cells (MSCs) into lung cells, A step of sowing MSCs on a substrate; and A process of exposing a substrate seeded with MSCs to a growth medium containing retinoic acid and at least one human epidermal growth factor, thereby differentiating the MSCs into lung cells that express at least one epithelial marker. The method, including the method. [Invention 1002] The method of the present invention 1001, wherein the MSC is selected from the group consisting of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs). [Invention 1003] The method of the present invention 1001, wherein lung cells exhibit at least one characteristic of type II alveolar epithelial cells. [Invention 1004] The method of the present invention 1003, wherein at least one feature of type II alveolar epithelial cells is the expression of at least one epithelial marker selected from the group consisting of proSPC and cytokeratin-5. [Invention 1005] The method of the present invention 1001, wherein lung cells exhibit at least one characteristic of Clara cells. [Invention 1006] The method of the present invention 1005, wherein at least one characteristic of Clara cells is the expression of Clara cell secreted protein (CCSP). [Invention 1007] The method of the present invention 1001, wherein the substrate is decellularized lung tissue. [Invention 1008] The method of the present invention 1001, wherein the substrate is a coating containing an extracellular matrix. [Invention 1009] The method of the present invention 1008, wherein the extracellular matrix comprises one or more of human ECM, laminin, fibronectin, collagen IV, and collagen I. [Invention 1010] A population of lung epithelial cells differentiated from mesenchymal stem cells (MSCs) that express at least one epithelial marker selected from the group consisting of CCSP, proSPC, and cytokeratin-5. [Invention 1011] A population of lung cells according to the present invention 1010, wherein the MSCs are selected from the group consisting of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs). [Invention 1012] A population of lung cells according to the present invention 1010, wherein the lung epithelial cells are selected from the group consisting of type I alveolar epithelial cells, type II alveolar epithelial cells, and Clara cells. [Invention 1013] A population of lung cells according to the present invention 1010, wherein lung epithelial cells are seeded on a substrate. [Invention 1014] A population of lung cells according to the present invention 1013, wherein the substrate is decellularized lung tissue. [Invention 1015] A population of lung cells according to the present invention 1013, wherein the substrate is a coating containing an extracellular matrix. [Invention 1016] A population of lung cells according to the present invention 1015, wherein the extracellular matrix comprises one or more of human ECM, laminin, fibronectin, collagen IV, and collagen I. [Invention 1017] A population of lung cells according to the present invention 1010, including genetically modified cells. [Invention 1018] A population of lung cells according to the present invention 1017, wherein the genetically modified cells are lung epithelial cells that have been genetically modified to express a therapeutic gene. [Invention 1019] A method for reducing or treating a lung abnormality in a mammal, comprising the step of administering a therapeutically effective dose to a mammal of a population of lung epithelial cells differentiated from mesenchymal stem cells (MSCs), wherein the lung epithelial cells express at least one epithelial marker selected from the group consisting of CCSP, proSPC, and cytokeratin-5. [Invention 1020] A method for regulating the differentiation of mesenchymal stem cells (MSCs) into lung cells, comprising the step of culturing MSCs on a substrate to differentiate the MSCs into lung cells. [Invention 1021] The method of the present invention 1020, wherein, when the MSCs are bone marrow-derived MSCs (BM-MSCs), the MSCs differentiate into cells exhibiting at least one characteristic of type II alveolar epithelial cells. [Invention 1022] The method of the present invention 1021, wherein at least one feature of type II alveolar epithelial cells is the expression of at least one selected from the group consisting of proSPC and cytokeratin-5. [Invention 1023] The method of the present invention 1020, wherein, when the MSCs are adipose tissue-derived MSCs (AT-MSCs), the MSCs differentiate into cells exhibiting at least one characteristic of Clara cells. [Invention 1024] The method of the present invention 1023, wherein at least one characteristic of the Clara cells is the expression of Clara cell secreted protein (CCSP). [Invention 1025] A population of lung cells produced by a method for differentiating mesenchymal stem cells (MSCs) into lung cells, which includes the step of culturing MSCs on a substrate and thereby differentiating the MSCs into lung cells. [Invention 1026] A population of lung cells according to the present invention 1025, wherein the MSCs are selected from the group consisting of bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs). [Invention 1027] A population of lung cells according to the present invention 1025, including cells genetically modified to express therapeutic genes. [Invention 1028] A method for reducing or treating lung abnormalities in a mammal, comprising the step of administering a therapeutically effective dose to a mammal a population of lung cells produced by a method for differentiating mesenchymal stem cells (MSCs) into lung cells, wherein the differentiation method includes culturing MSCs on a substrate and thereby differentiating MSCs into lung cells. [Brief explanation of the drawing]

[0021] The following detailed description of preferred embodiments of the present invention will be better understood in conjunction with the accompanying drawings. Preferred embodiments are shown in the drawings to illustrate the present invention. However, it should be understood that the present invention is not limited to the exact configuration and means of the illustrated embodiments.

[0022] [Figure 1] Figure 1, including Figures 1A-1F, depicts the results of FACS analysis of adherent, passage 2 hBM-MSC and hAT-MSC samples, suggesting that the cells express mesenchymal stromal cell markers and epithelial markers. (Figure 1A) Morphology of passage 2 hBM-MSCs. (Figure 1B) hBM-MSCs are positive for MSC markers including CD90, CD105, and CD73, and they are CD45 negative. (Figure 1C) A subpopulation of hBM-MSCs expresses the epithelial markers proSPC, CCSP, and cytokeratin-5. (Figure 1D) Morphology of passage 2 hAT-MSCs. (Figure 1E) FACS analysis of hAT-MSCs confirms that these cells are also positive for MSC markers including CD90, CD105, and CD73, and they are CD45 negative. (Figure 1F) hAT-MSCs also include a population of cells positive for proSPC, CCSP, and cytokeratin-5. Isotype controls and experimental samples are shown. [Figure 2]Figure 2, including Figures 2A-2F: Depicts the results of experiments investigating lung bioreactor cultures seeded with hBM-MSCs. (Figures 2A, 2B) DAPI staining is used to reveal the nucleus in native lung (Figure 2A) and the anucleation in decellularized lung (Figure B). (Figure 2C) H&E tissue sections of lung cultured for 7 days in SAGM after seeding with hBM-MSCs. (Figure 2D) Immunostaining for pro-SPC shows numerous cells positive for type 2 lung cell markers. (Figures 2E, 2F) Immunostaining revealed no cells positive for the Clara cell marker CCSP (Figure 2E), but a small number of cells positive for cytokeratin-5 were present (Figure 2F). (Figures 2G, 2H) TEM analysis was performed as an additional method to actively identify hBM-MSC-derived cells as type 2 lung cells. TEM analysis of native type 2 cells (Figure 2E) (arrows indicate laminae; chevron symbols indicate secretory vesicles). (Figure 2F) Lungs re-seeded with hBM-MSCs contain cells that have both laminae (arrows) and secretory vesicles (arrowheads), which are characteristic of type 2 lung cells. [Figure 3] Figure 3, including Figures 3A-3D, illustrates the results of an experiment demonstrating that human adipose tissue mesenchymal stromal cells (hAT-MSCs), when cultured in a lung bioreactor, give rise to type II lung cell-like cells and Clara-like cells that line the airways. (Figure 3A) hAT-MSCs firmly rearrange the lung matrix after 7 days of culture in SAGM within a cell-free rat lung bioreactor. H&E staining reveals specific affinities for the cells to inhabit the airway lining (arrows). (Figure 3B) Cells lining the airways (arrows) are positive for the Clara cell marker CCSP. (Figure 3C) Cells proliferating on the matrix are positive for pro-SPC. The inset shows granular cytoplasmic staining of pro-SPC-positive cells. (Figure 3D) There is no indication that hAT-MSCs maintain cytokeratin-5 expression when proliferated on a decellularized matrix. [Figure 4]Figure 4, including Figures 4A-4G: Depicts experimental results demonstrating RT-PCR analysis of lung bioreactor cultures seeded with either hBM-MSCs or hAT-MSCs at days 3 and 7. (Figures 4A-C) hBM-MSCs increased gene expression levels of distal epithelial genes, including SPC, aquaporin-1, and caveolin-1, over time in lung bioreactor cultures. (Figures 4D-F) Similarly, hAT-MSCs increased distal gene expression with longer culture periods. All cell doublings in the lung bioreactors are compared to MSCs grown in tissue culture flasks. (Figure 4G) Primers used to amplify gene sequences. [Figure 5] Figure 5, including Figures 5A-5E, illustrates the results of experiments demonstrating that hAT-MSCs and hBM-MSCs grown in SAGM actively produce surfactant. RT-PCR analysis of BM-MSCs (Figure 5A) and hAT-MSCs (Figure 5B) seeded on cell-free rat lungs suggests a gradual increase in SPC expression over time under culture conditions. hBM-MSCs (Figure 5D) and hAT-MSCs (Figure 5E) cultured in SAGM for 7 days in lung slices exhibit visible surfactant droplets in the culture medium in contact with the lung slices. (Figure 5C) ELISA of the culture medium samples suggests that at day 3, 5.5 ng / mL of SPC was present in hAT-MSC lung slice cultures and 3.3 ng / mL in hBM-MSC cultures. On day 7, the SPC concentration was even lower, at 1.1 ng / mL for hAT-MSCs and 0.26 ng / mL for hBM-MSCs. All ELISA values ​​were normalized for SAGM medium only. The bars represent the standard error (SEM). [Figure 6]Figure 6, including Figures 6A-6F, is a series of images demonstrating that substrate coatings affect the expression of epithelial markers in hBM-MSCs and hAT-MSCs. hBM-MSCs and hAT-MSCs were cultured in SAGM medium on various ECM coatings (human ECM, laminin, fibronectin, collagen IV, and collagen I). After 7 days of culture, hBM-MSCs (Figure 6A) and hAT-MSCs (Figure 6B) were analyzed by FACS for the expression of CCSP, pro-SPC, and cytokeratin 5. Both MSC sources differed in terms of epithelial marker expression when cultured on different surface coatings (Figures 6C, 6D). RT-PCR analysis was similarly performed on the expression of CCSP, SPC, and cytokeratin 5 after culture on various surface coatings. The RT-PCR data were in good agreement with the FACS analysis, suggesting that the highest SPC expression was observed in MSCs grown in human ECM compared to MSCs grown on other surface coatings. Furthermore, these experiments revealed differences in cell morphology between hBM-MSCs (Figure 6E) and hAT-MSCs (Figure 6F) grown on human ECM. [Figure 7]Figure 7, including Figures 7A-7F, illustrates the results of an experiment demonstrating that rat lung bioreactor cultures seeded with hBM-MSCs and grown in 10% FBS / DMEM possess adherent cells with fibroblast-like morphology and protein expression. (Figure 7A) H&E tissue images of 7-day cultures demonstrate the presence of cells uniformly distributed throughout the recellularized organ. The adherent cells are mostly fibroblast-like in morphology. (Figure 7B) Immunofluorescence for α-SMA, a marker used to detect myofibroblasts, reveals that the majority of cells are positive for the myofibroblast marker. (Figures 7C, 7D) Immunofluorescence analysis of CCSP expression in cytospins of hBM-MSCs grown in either 10% FBS / DMEM or small airway growth medium (SAGM). CCSP expression is maintained in cells cultured under both conditions. (Figures 7E, 7F) Immunofluorescence of a-sma expression in cells grown in either medium shows that a-sma is almost absent when cells are grown in SAGM. [Figure 8] Figure 8, including Figures 8A-8D, illustrates the results of an experiment demonstrating the absence of visible surfactant secretion in lung slices reseeded with either hAT-MSCs or hBM-MSCs and cultured in 10% FBS / DMEM. (Figure 8A) hAT-MSCs and (Figure 8B) hBM-MSCs cultured on lung slices in 10% FBS / DMEM did not contain visible surfactant in the culture medium. (Figures 8C, 8D) As a control, unseeded samples were also analyzed for the surfactant pool in the culture medium, and these also contained no visible surfactant. [Figure 9] These are a series of FACS plots of hBM-MSCs grown in SAGM for 7 days on various ECM substrates in tissue culture flasks. [Figure 10] These are a series of FACS plots of hAT-MSCs grown in SAGM for 7 days on various ECM substrates in tissue culture flasks. [Figure 11]Figure 11, including Figures 11A-11I, illustrates the results of an experiment demonstrating that hBM-MSCs cultured on a decellularized liver matrix are morphologically different from those cultured on the lung. (Figure 11A) Whole image of a decellularized liver. (Figure 11B) H&E tissue image of a decellularized liver clearly shows that there are no residual cells on the matrix. (Figures 11C, 11D) Re-seeded liver slices were cultured for 3 days in 10% FBS / DMEM (Figure 11C) or SAGM (Figure 11D). A morphological comparison between these cells and cells seeded in the lung (Figure 3C) shows significant differences in cell shape. Cells grown in either 10% FBS / DMEM or SAGM were negative for CCSP and cytokeratin 5 (Figures 11E, 11G, 11H, 11J), while very few cells remained positive for proSPC (Figures 11F, 11I). [Figure 12] This is a panel of graphs showing whole, unfractionated human bone marrow cells as a negative staining control for lung epithelial markers. Unfractionated human bone marrow mononuclear cells were stained for the lung epithelial markers proSPC, CCSP, and cytokeratin-5. No indication of unfractionated cells expressing positive markers was observed. Shaded peaks on the right indicate cells without antibody control; peaks on the left indicate cells incubated with antibody. [Modes for carrying out the invention]

[0023] Detailed explanation This invention is based on the discovery that mesenchymal stromal cells derived from human bone marrow and adipose tissue (hBM-MSC and hAT-MSC, respectively) undergo changes in lung epithelial marker expression depending on the culture substrate (e.g., decellularized lung tissue or culture plates containing various surface coatings).

[0024] In one embodiment, when cultured on decellularized lung tissue, hBM-MSCs adhere to the decellularized lung matrix, particularly in the peripheral lung region, express type 2 lung cell-associated markers (pro-SPC), contain laminae, and actively secrete surfactant protein C. hBM-MSCs also differentiate into cells that are positive for cytokeratin-5 but negative for other lung-associated markers such as CCSP.

[0025] In one embodiment, hAT-MSCs, when cultured on decellularized lung tissue, exhibited type 2 lung cell-like and Clara-like cellular features. In contrast to hBM-MSCs, hAT-MSCs produce Clara-like cells (e.g., CCSP-positive) that line the airways at anatomically accurate locations. Furthermore, in contrast to hBM-MSCs, hAT-MSCs do not produce cytokeratin-5-positive cells.

[0026] Therefore, the present invention is based on the discovery that the ability of MSCs to differentiate toward the lung epithelial phenotype depends on the substrate, primary tissue, and culture medium.

[0027] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. Generally, the terminology used herein, as well as experimental procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization, are well known and commonly used in the art.

[0028] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one or more elements.

[0029] The term "approximately" will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used.

[0030] The term “adult stem cells” or “ASC” is used to refer to any multipotent stem cells derived from non-embryonic tissues, including fetal, juvenile, and adult tissues. Stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, fat, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As described above, stem cells have been found to be present in virtually any tissue. Therefore, the present invention understands that stem cell populations can be isolated from virtually any animal tissue.

[0031] As used herein, "autologous" means biological material that originates from the same individual from which the material will later be reintroduced.

[0032] As used herein, "homogeneous" means biological material derived from a genetically different individual of the same species as the individual into which the material will be introduced.

[0033] As used herein, “mitigating” a disease, abnormality, disorder, or condition means reducing the severity of one or more symptoms of that disease, abnormality, disorder, or condition.

[0034] As used herein, the term “basal medium” refers to a solution of amino acids, vitamins, salts, and nutrients that is effective in supporting the growth of cultured cells, although these compounds will not typically support cell growth unless supplemented with additional compounds. Nutrients include carbon sources that cells can metabolize (e.g., sugars such as glucose), as well as other compounds necessary for cell survival. These are compounds that cells cannot synthesize themselves because one or more genes encoding the proteins necessary to synthesize those compounds (e.g., essential amino acids) are absent, or, with respect to compounds that cells can synthesize, these are compounds in which the genes encoding the necessary biosynthetic proteins are not expressed at sufficient levels due to a specific developmental stage of the cell. Although several basal media are known in the field of mammalian cell culture, such as Dulbecco's Modified Eagle Media (DMEM), Knockout-DMEM (KO-DMEM), and DMEM / F12, any basal medium that supports the growth of primate embryonic stem cells in a substantially undifferentiated state can be used.

[0035] As used herein, "biocompatible" means any material that, when implanted in a mammal, does not induce an adverse response in that mammal. Biocompatible materials, when introduced into an organism, are not toxic or harmful to that organism and do not induce immunological rejection of the material in that mammal.

[0036] As used herein, the term “biocompatible lattice” is intended to mean a substrate capable of promoting the formation of three-dimensional structures that facilitate tissue development. Therefore, for example, cells can be cultured or seeded on such a biocompatible lattice, which may contain extracellular matrix material, synthetic polymers, cytokines, growth factors, etc. The lattice can be molded into a desired shape to promote the development of tissue types. Furthermore, at least in the early stages of cell culture, the culture medium and / or substrate may be supplemented with factors that promote the development of appropriate tissue types and structures (e.g., growth factors, cytokines, extracellular matrix material, etc.).

[0037] As used herein, “bioactive agent” may include one or more of the following: chemotactic substances; therapeutic agents (e.g., antibiotics, steroidal and nonsteroidal analgesics and anti-inflammatory drugs (including certain amino acids such as glycine), immunosuppressants and anticancer drugs); various proteins (e.g., short-term peptides, bone morphogenetic proteins, collagen, hyaluronic acid, glycoproteins, and lipoproteins); cell adhesion mediators; biologically active ligands; integrin-binding sequences; ligands; various growth agents and / or differentiation agents and their fragments (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (e.g., bFGF), platelet-derived growth factor (PDGF), insulin-derived growth factors (e.g., IGF-1, IGF-II), and transformation growth factors (e.g., TGFβ) I-III), parathyroid hormone, parathyroid hormone-related peptides, bone morphogenetic proteins (e.g., BMP-2, BMP-4; BMP-6; BMP-7; BMP-12; BMP-13; BMP-14), sonic hedgehog, growth and differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52, and MP-52 variant rhGDF-5), cartilage-derived morphogenetic proteins (CDMP-1; CDMP-2, CDMP-3)); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboelastin; thrombin-derived peptides; heparin-binding domains; heparin; heparan sulfate. Suitable effectors also include agonists and antagonists of the above agents. Growth factors can also include combinations of the growth factors mentioned above. In addition, growth factors can be autologous growth factors supplied by platelets in the blood. In this case, platelet-derived growth factors would be an indeterminate cocktail of various growth factors.If other such substances have therapeutic value in the field of orthopedics, it is expected that at least some of these substances will have use in the present invention, and such substances should be included in the meaning of “bioactive agent” and “bioactive agents” unless otherwise expressly limited. Preferred examples of bioactive agents include culture media, bone morphogenetic proteins, growth factors, growth and differentiation factors, recombinant human growth factors, cartilage-derived morphogenetic proteins, hydrogels, polymers, antibiotics, anti-inflammatory drugs, immunosuppressants, autologous cells, allogeneic or xenologous cells, such as stem cells, chondrocytes, fibroblasts, and proteins, such as collagen and hyaluronic acid. Bioactive agents may be autologous, allogeneic, xenogenic, or recombinant.

[0038] The terms “biologically compatible carrier” or “biologically compatible culture medium” refer to reagents, cells, compounds, materials, compositions, and / or dosage formulations suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other complications, in proportion to a reasonable benefit-benefit ratio.

[0039] The terms "cell" and "population of cells" are used interchangeably and refer to multiple cells, i.e., two or more cells. This population may be a pure population containing one cell type, or it may contain two or more cell types. In this invention, there is no limit to the number of cell types that a population of cells may contain.

[0040] As used herein, the term “cell medium” refers to a medium useful for culturing cells. An example of a cell medium is a medium containing DMEM / F 12 Ham's, 10% fetal bovine serum, 100 U penicillin / streptomycin 100 μg / fungizone 0.25 μg. Typically, a cell medium includes a basal medium, serum, and antibiotics / antifungal agents. However, cells may be cultured in an antibiotic / antifungal stromal cell medium, supplemented with at least one growth factor. Preferably, the growth factor is human epidermal growth factor (hEGF). The preferred concentration of hEGF is about 1–50 ng / ml, more preferably about 5 ng / ml. The preferred basal medium is DMEM / F 12 (1:1). The preferred serum is fetal bovine serum (FBS), however other serums may be used, including horse serum or human serum. Preferably, up to 20% FBS may be added to the culture medium to support the proliferation of stromal cells. However, standard media may be used if the necessary growth factors, cytokines, and hormones in FBS for cell proliferation are identified and provided in appropriate concentrations in the growth medium. It is further recognized that further components may be added to the culture medium. Such components include, but are not limited to, antibiotics, antifungal agents, albumin, growth factors, amino acids, and other components known to the art for cell culture. Antibiotics that may be added to the culture medium include, but are not limited to, penicillin and streptomycin. The concentration of penicillin in the culture medium is about 10 to about 200 units per ml. The concentration of streptomycin in the culture medium is about 10 to about 200 μg / ml. However, the present invention should never be construed as being limited to any one medium for culturing cells. Rather, any medium capable of supporting cells in tissue culture may be used.

[0041] As used herein, the terms “decellularized” or “decellularized” refer to a biological structure (e.g., an organ or part of an organ) from which the cellular and tissue contents have been removed, leaving an intact, cell-free basic structure. Organs, such as the kidney, are composed of various specialized tissues. Specialized organ tissue structures, or parenchyma, provide specific functions associated with the organ. The interstitium is the network of supporting fibers of an organ. Most organs have an interstitial framework composed of non-specialized connective tissue supporting specialized tissues. The decellularization process removes specialized tissues, leaving a complex three-dimensional network of connective tissue. The basic structure of connective tissue is primarily composed of collagen. Decellularized structures become biocompatible substrates into which different cell populations can be injected. Decellularized biological structures can be rigid or semi-rigid and may have the ability to change their shape. Examples of decellularized organs useful in the present invention include, but are not limited to, the heart, lungs, kidneys, liver, pancreas, spleen, bladder, ureters and urethra, cartilage, bone, brain, spinal cord, and peripheral nerves.

[0042] As used herein, the term “dedifferentiation” refers to a cell returning to a less specialized state. After dedifferentiation, such cells will have the ability to differentiate into more or different cell types than was possible before reprogramming. The process of reverse differentiation (i.e., dedifferentiation) is likely to be more complex than differentiation and requires “reprogramming” the cell to become more primitive.

[0043] The term “differentiated cell” refers to any primary cell that, in its natural form, is not pluripotent as the term is defined herein. In other words, the term “differentiated cell” refers to a more specialized cell type derived from a less specialized cell type (e.g., stem cells such as induced pluripotent stem cells) in the process of cell differentiation. While not wishing to be limited to theory, pluripotent stem cells during normal individual development can initially differentiate into endodermal cells and other endodermal cell types capable of forming lung cells. Endoderm cells can also differentiate into other cells of endoderm origin, such as those of the lungs, liver, intestines, and thymus.

[0044] In this specification, “differentiation medium” is used to mean a cell growth medium that contains or lacks additives such that, when fully differentiated, stem cells, fetal lung cells, or other such progenitor cells are incubated in the medium, they develop into cells having some or all of the characteristics of differentiated cells.

[0045] The term “embryonic stem cells” is used to refer to pluripotent stem cells in the inner cell mass of a blastocyst (see U.S. Patents No. 5,843,780 and 6,200,806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Patents No. 5,945,577, 5,994,619 and 6,235,970). Distinctive features of embryonic stem cells define the embryonic stem cell phenotype. Thus, cells have the embryonic stem cell phenotype if they possess one or more features specific to embryonic stem cells that can distinguish them from other cells. Exemplary distinctive features of embryonic stem cells include, but are not limited to, gene expression profiles, proliferative capacity, differentiation capacity, karyotype, and responsiveness to specific culture conditions.

[0046] As used herein, “epithelial cells” means cells that form the outer surface of the body and line the surfaces of organs, lumens, and mucous membranes.

[0047] As used herein, “endothelial cells” means the cells that line blood vessels and lymphatic vessels, as well as various other body cavities.

[0048] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, or system.

[0049] "Exogenous" refers to any material that is introduced into or produced outside of an organism, cell, or system.

[0050] "Code" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which is that it serves as a template for the synthesis in a biological process of other polymers and macromolecules having a specific sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a specific sequence of amino acids and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a certain gene produces a certain protein within a cell or other biological system, then that gene codes for that protein. Both the coding strand, which has the same nucleotide sequence as the mRNA sequence and is usually listed in sequence listings, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be said to code for a protein, or other products of that gene or cDNA.

[0051] As used herein, the term “endodermal cell” refers to a cell derived from one of the three primary germ cell layers in the very early stages of the embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endoderm cells differentiate to first give rise to the embryonic gastrointestinal tract, then to the respiratory tract and the inner surface of the digestive tract (e.g., the intestines), the liver, and the pancreas.

[0052] In this specification, "expandability" is used to refer to the ability of cells to proliferate, for example, to increase in number, or, in the case of a group of cells, to undergo population doubling.

[0053] As used herein, “extracellular matrix composition” comprises both or any portion thereof of a soluble fraction and an insoluble fraction. The insoluble fraction comprises secreted ECM proteins and biological components deposited on a support or scaffold. The soluble fraction comprises the medium in which the cells were cultured and in which the cells secreted active substances, and comprises those proteins and biological components that have not been deposited on a scaffold. Both fractions may be recovered, optionally further processed, and used separately or together in the various uses described herein.

[0054] "Fibrosis" is the formation or development of excessive fibrous connective tissue in an organ or tissue as a repair or reaction process, in contrast to the formation of fibrous tissue as a normal component of the organ or tissue. Pulmonary fibrosis is a severe chronic disease characterized by loss of elasticity, replacement of pulmonary epithelial cells with interstitial myofibroblasts, and accumulation of extracellular matrix proteins in the pulmonary interstitium, resulting in a remodeling of the lung structure.

[0055] As used herein, “graft” means a cell, tissue, or organ that is typically implanted in an individual to replace, correct, or otherwise overcome an abnormality. A graft may further include a scaffold. The tissue or organ may consist of cells derived from the same individual, and such a graft is referred to herein by the following interchangeable terms: “autograft,” “autologous transplant,” “autologous implant,” and “autograft.” A graft containing cells derived from genetically different individuals of the same species is referred to herein by the following interchangeable terms: “allograft,” “allogeneic transplant,” “allogeneic implant,” and “allogeneic graft.” A graft obtained from identical twins, a brother and sister, of the same individual is referred to herein by the following interchangeable terms: “isograft,” “allogeneic transplant,” “allogeneic implant,” or “allogeneic graft.” "Xenograft," "external transplant," or "external implant" refers to the transfer of a graft from one individual to another individual of a different species.

[0056] As used herein, the term “growth factor product” means a protein, peptide, mitogen, or other molecule that has a growth effect, proliferation effect, differentiation effect, or nutritional effect on cells. Growth factors include, but are not limited to, fibroblast growth factor (FGF), basic fibroblast growth factor (bFGF), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), insulin-like growth factor I (IGF-T), insulin-like growth factor II (IGF-II), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), activin A, bone morphogenetic protein (BMP), insulin, growth hormone, erythropoietin, thrombopoietin, interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), macrophage colony-stimulating factor, c-kit ligand / stem cell factor, osteoprotegerin ligand, insulin, nerve growth factor, ciliary neurotrophic factor, cytokines, chemokines, morphogens, neutralizing antibodies, other proteins, and small molecules. Preferably, the FGF is selected from the group consisting of FGF2, FGF7, FGF10, and any combination thereof.

[0057] As used herein, the term “growth medium” is intended to mean a culture medium that promotes cell growth. Growth media will generally contain animal serum. In some cases, growth media may not contain animal serum.

[0058] "Isolated cells" refers to cells isolated from other components and / or cells naturally associated with them in a tissue or mammal.

[0059] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from its naturally occurring adjacent sequences, that is, a DNA fragment taken from sequences adjacent to the fragment (i.e., sequences adjacent to the fragment in its naturally occurring genome). This term also applies to nucleic acids substantially purified from other components naturally associated with that nucleic acid, i.e., RNA or DNA or proteins naturally associated with it in cells. Therefore, this term includes, for example, recombinant DNA that is incorporated into a vector, incorporated into an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a protist or eukaryote, or recombinant DNA that exists independently of other sequences as a separate molecule (i.e., cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0060] The term “lung-specific” refers to nucleic acid molecules or polypeptides that are predominantly expressed in the lungs compared to other tissues of the body. In a preferred embodiment, the “lung-specific” nucleic acid molecule or polypeptide is expressed at a level 5 times higher than in any other tissue of the body. In a more preferred embodiment, the “lung-specific” nucleic acid molecule or polypeptide is expressed at a level 10 times higher than in any other tissue of the body, and more preferably at a level at least 15 times, 20 times, 25 times, 50 times, or 100 times higher than in any other tissue of the body. Nucleic acid molecule levels may be measured by nucleic acid hybridization, such as Northern blot hybridization, or by quantitative PCR. Polypeptide levels may be measured by any method known to accurately measure protein levels, such as Western blot analysis.

[0061] "Lung tissue" includes, but is not limited to, all lung tissue structures and associated tissues, including, veins, arteries, blood vessels, capillaries, and cells of the type that are part of or associated with such structures; lung and pleural tissue; and may include, but is not limited to, vascular smooth muscle, pericytes, and vascular endothelial lineage and / or phenotypes.

[0062] The terms “precursor cell,” “progenitor cell,” and “stem cell” are interchangeable in the art and, as used herein, refer to pluripotent or lineage-uncommitted progenitor cells that potentially have the ability to regenerate themselves through mitosis indefinitely or to produce progeny cells that will differentiate into desired cell types. In contrast to pluripotent stem cells, lineage-committed progenitor cells are generally not considered to have the ability to produce a number of cell types with distinct phenotypes. Instead, progenitor cells produce one or possibly two lineage-committed cell types.

[0063] "Proliferation" is used herein to mean the replication or multiplication of similar forms, particularly of cells. That is, proliferation encompasses the production of a larger number of cells, for example, simply counting the number of cells. 3 This can be measured by, for example, measuring the uptake of H-thymidine into cells.

[0064] "Cell cycle progression or progression through the cell cycle" is used herein as to mean the process by which a cell prepares for and / or enters mitosis and / or meiosis. Progression through the cell cycle includes progression through the G1, S, G2, and M phases.

[0065] As used herein, “scaffold” means a structure comprising biocompatible material that provides a surface suitable for cell adhesion and proliferation. A scaffold may also provide mechanical stability and mechanical support. A scaffold may take on a specific shape or form so as to influence or define the boundaries of a three-dimensional shape, or it may take on the form exhibited by a population of proliferating cells. Such shapes or forms include, but are not limited to, thin films (e.g., forms with two dimensions substantially larger than the third dimension), ribbons, strings, sheets, flat disks, cylinders, spheres, and amorphous three-dimensional shapes.

[0066] As used herein, the terms "small airway growth medium" or "SAGM" refer to a growth medium containing one or more of the following components: Hydrocortisone, epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, and fatty acid-free bovine serum albumin.

[0067] As used herein, the term “stem cell” refers to both the earliest regenerative cell populations involved in giving rise to cell aggregates in tissues or bodies, and very early progenitor cells that are somewhat more differentiated, but nevertheless not constrained by differentiation, and can easily revert to becoming part of the earliest regenerative cell population.

[0068] As used herein, “substantially purified” cells are cells that are essentially free of other cell types. Therefore, substantially purified cells are those that have been purified from other cell types that are normally associated with them in their natural state.

[0069] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, the subject is preferably a mammal, such as non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys and humans), most preferably humans.

[0070] As used herein, “to treat” means to reduce the frequency with which a patient experiences symptoms of a disease, abnormality, disorder, or adverse condition.

[0071] As used herein, “therapeutic effective dose” is the amount of the composition of the present invention that is sufficient to give a beneficial effect to the individual to whom the composition is administered.

[0072] As used herein, “tissue engineering” refers to the process of generating tissue ex vivo for use in tissue replacement or reconstruction. Tissue engineering is an example of “regenerative medicine,” which encompasses efforts to repair or replace tissues and organs using the incorporation of cells, genes or other biological components and biotechnological materials and techniques.

[0073] As used herein, the terms "tissue grafting" and "tissue reconstruction" both refer to the implantation of grafts into an organism to treat or alleviate tissue abnormalities, such as lung abnormalities or soft tissue abnormalities.

[0074] A "graft" refers to a biocompatible lattice or donor tissue, organ, or cells intended for transplantation. Examples of grafts include, but are not limited to, skin cells or tissue, bone marrow, and parenchymal organs such as the heart, pancreas, kidneys, lungs, and liver.

[0075] Generally, "nutritional factors" are defined as substances that promote cell survival, growth, proliferation, and / or maturation, and that stimulate increased cell activity.

[0076] Scope: Throughout this disclosure, various aspects of the invention can be presented in the form of scope. It should be understood that descriptions in the form of scope are merely for convenience and simplification and should not be interpreted as inflexible limitations on the scope of the invention. Accordingly, descriptions of scope should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0077] explanation This invention relates to the discovery that various stem cell types (e.g., bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs)) undergo significant changes in lung epithelial marker expression depending on their culture substrate. Examples of substrates include, but are not limited to, decellularized lung tissue and culture plates containing various surface coatings. Instructions for decellularized lung tissue can be found in U.S. Patent Application Publication No. 20120064050, which is incorporated herein by reference.

[0078] In one embodiment, BM-MSCs, when placed on decellularized lung tissue, may express the type II lung cell marker pro-SPC and the proximal airway marker cytokeratin-5. In one embodiment, BM-MSCs, when expanded and cultured under suitable conditions, may exhibit the phenotype of type II alveolar epithelial cells (also known as type II lung cells). In another embodiment, when expanded and cultured under suitable conditions, BM-MSCs exhibit function-related cytoplasmic structures similar to those contained within native type II cells.

[0079] In another embodiment, AT-MSCs produce pro-SPC positive cells. In another embodiment, when expanded and cultured under suitable conditions, AT-MSCs produce Clara-like cells (e.g., CCSP positive) that line the airways at anatomically precise locations. This is in contrast to BM-MSCs, which neither maintain CCSP expression nor adhere to the airways. In another embodiment, in contrast to BM-MSCs, AT-MSCs do not produce cytokeratin-5 positive cells.

[0080] Therefore, the present invention provides compositions and methods for producing desired cell types. Thus, the cells of the present invention are a promising source of cells for therapeutic use to treat peripheral lung diseases, lung injuries, and genetic disorders affecting the lungs.

[0081] The present invention provides a method for differentiating BM-MSCs into cells exhibiting the type II lung cell marker pro-SPC and the proximal airway marker cytokeratin-5. In one embodiment, the present invention provides a method for differentiating BM-MSCs to exhibit at least one phenotype of type II alveolar epithelial cells. In another embodiment, the present invention provides a method for differentiating BM-MSCs to exhibit function-related cytoplasmic structures similar to those contained within native type II cells.

[0082] Therefore, the present invention provides a method for inducing differentiation of BM-MSCs into cell types and tissues associated with type II alveolar epithelial cells.

[0083] The present invention provides a method for differentiating AT-MSCs into cells that exhibit the pro-SPC type 2 lung cell marker but do not produce cytokeratin-5 positive cells. In one embodiment, the present invention provides a method for differentiating AT-MSCs into cells that are positive for the Clara cell marker CCSP. In another embodiment, the present invention provides a method for differentiating AT-MSCs to produce Clara-like cells (e.g., CCSP positive) that line the airways at anatomically precise locations.

[0084] Therefore, the present invention provides a method for inducing the differentiation of AT-MSCs into cell types and tissues associated with Clara cells.

[0085] Culture conditions The present invention relates to the use of any cells to differentiate into a type of lung cell (e.g., type II alveolar epithelial cells or Clara cells). Preferably, the appropriate cells or cell populations are regenerative. Examples of regenerative cells include, but are not limited to, stem cells, embryonic stem cells, adult stem cells, umbilical cord blood cells, tissue-derived stem cells or progenitor cells, bone marrow-derived stem cells or progenitor cells, blood-derived stem cells or progenitor cells, adipose tissue-derived stem cells or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, multipotent adult progenitor cells (MAPCs), fetal lung cells, differentiated lung epithelial cells, lung progenitor cells, vascular progenitor cells, differentiated vascular cells, etc. Further regenerative cells that can be used include bone marrow-derived stem cells, e.g., bone marrow mononuclear cells (BM-MNCs), endothelial or vascular stem cells or progenitor cells, and peripheral blood-derived stem cells, e.g., endothelial progenitor cells (EPCs).

[0086] Preferably, suitable cells are isolated from mammals, more preferably from primates, and even more preferably from humans. Cells useful in the method of the present invention are isolated using, for example, the methods discussed herein in the Examples section, or by any method known in the art. After isolation, suitable cells are cultured in a culture medium.

[0087] Cell culture medium compositions typically contain essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, and nucleosides. Cell culture media are intended to provide the necessary components to meet the nutritional requirements for cell growth in a controlled, artificial, and in vitro environment. Nutrient formulations, pH, and osmotic molar concentrations vary depending on parameters such as the cell type, cell density, and culture system used. Many cell culture medium formulations are documented in the literature, and several are commercially available.

[0088] Once a culture medium is incubated with cells, it becomes known to those skilled in the art as a "conditioned medium." The conditioned medium contains many of the original components of the medium, along with various cellular metabolites and secreted proteins, such as bioactive growth factors, inflammatory mediators, and other extracellular proteins.

[0089] Those skilled in the art will recognize that culture conditions can be modified to suit the appropriate cells. Medium formulations supporting MSC growth include Eagle's Minimum Essential Medium, ADC-1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ medium (with and without Fitton-Jackson's modification), and Eagle's basal medium (BME-Earl's salt base (salt) This includes, but is not limited to, Eagle Medium (with added base), Dulbecco's Modified Eagle Medium (DMEM-serum-free), Yamane, IMEM-20, Glasgow Modified Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, M199 Medium (M199E-containing Earl's salt base), M199 Medium (M199H-containing Hanks' salt base), Eagle Minimal Essential Medium (MEM-E-containing Earl's salt base), Eagle Minimal Essential Medium (MEM-H-containing Hanks' salt base), and Eagle Minimal Essential Medium (MEM-NAA containing non-essential amino acids).

[0090] In further non-limiting examples of culture media useful in the method of the present invention, fetal serum of a bovine or other species may be contained at a concentration of at least 1% to about 30%, preferably at least about 5% to 15%, and most preferably about 10%. Fetal extract of a bovine or other species may be present at a concentration of about 1% to 30%, preferably at least about 5% to 15%, and most preferably about 10%.

[0091] Typically, the culture medium comprises a basal medium, serum, and antibiotics / antifungal agents. One preferred basal medium is DMEM / F12 (1:1). The preferred serum is fetal bovine serum (FBS), however other serums may be used, including horse serum or human serum. Preferably, up to 20% FBS will be added to the above medium to support the growth of MSCs. However, a standard medium can be used if the necessary growth factors, cytokines, and hormones in FBS for MSC growth are identified and provided in the growth medium at appropriate concentrations. It is further recognized that additional components may be added to the medium. Such components include, but are not limited to, antibiotics, antifungal agents, albumin, growth factors, amino acids, and other components known to the art for cell culture. Antibiotics that can be added to the medium include, but are not limited to, penicillin and streptomycin. The concentration of penicillin in the medium is about 10 to about 200 units per ml. The concentration of streptomycin in the culture medium is approximately 10 to 200 μg / ml. However, the present invention should not be construed as being limited to any one medium for culturing NPCs. Rather, any medium capable of supporting lung cells in tissue culture may be used.

[0092] Other components commonly used in culture medium formulations include fat-soluble vitamins (including A, D, E, and K), steroids and their derivatives, cholesterol, fatty acids and lipids Tween 80, 2-mercaptoethanolpyrimidines (pyramidines), as well as various adjuvants including serum (fetus, horse, calf, etc.), proteins (insulin, transferrin, growth factors, hormones, etc.), antibiotics (gentamicin, penicillin, streptomycin, amphotericin B, etc.), whole egg ultrafiltration solution, and adhesion factors (fibronectin, vitronectin, collagen, laminin, tenascin, etc.).

[0093] The culture medium may or may not need to be supplemented with growth factors and other proteins, such as adhesion molecules, because many of the cell constructs described in this application, particularly three-dimensional cell and tissue culture constructs, themselves produce such growth factors, adhesion molecules, and other products into the culture medium.

[0094] Soluble factors are factors that are released into and present in the culture medium of stem cells. Examples of soluble factors include, but are not limited to, vascular endothelial growth factor (VEGF), insulin growth factor (IGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and other members of the fibroblast growth factor family.

[0095] Following isolation, MSCs can be incubated in culture medium in a culture apparatus for a certain period, or until the cells become dense, before being subculturing in another culture apparatus. Following the initial plating, the cells can be cultured for approximately 6 days to obtain a subgeneration 0 (P0) population. The cells can be subculturished indefinitely, with each subgeneration involving culturing the cells for approximately 6–7 days, during which the cell doubling time can range from approximately 3–5 days. The culture apparatus can be any culture apparatus commonly used when culturing cells in vitro.

[0096] MSCs as described herein may be cryopreserved according to conventional methods. Preferably, about 1 million to 10 million cells are cryopreserved in a medium containing 10% DMSO in the gas phase of liquid N2. The frozen cells may be thawed by swirling in a 37°C water bath, resuspended in fresh growth medium, and expanded as described above.

[0097] The present invention also provides cells that can be "seeded" onto a scaffold. MSCs can be cultured on the scaffold. Cells can also be differentiated in vitro by culturing them in differentiation medium. Alternatively, cells can be differentiated in vivo if they establish contact with tissue within a mammal, or if they are close enough to the tissue to be affected by substances released from the tissue (e.g., growth factors, enzymes, or hormones). In other words, MSCs in a matrix can establish contact with tissues such as lungs by receiving signals from the tissue. Such signaling would occur, for example, when receptors on the surface of an MSC or on the surface of a cell derived from an MSC bind to molecules such as growth factors, enzymes, or hormones released by tissue within a mammal, and transmit signals from those molecules. Since these agents induce differentiation, the MSCs will come to express some, if not all, of the same proteins that are normally expressed by differentiated cells in the tissue to which they are placed.

[0098] Alternatively, or furthermore, the MSCs in the matrix can be induced to differentiate by adding a substance (e.g., a growth factor, enzyme, hormone, or other signaling molecule) to their cellular environment. For example, a substance can be added to the biological scaffold of the present invention.

[0099] In another embodiment, the present invention provides a method for culturing the cells of the present invention in a suitable growth medium in the presence of surface (e.g., a two-dimensional or three-dimensional surface) ECM proteins. In one embodiment, the ECM is coated onto the surface of the culture apparatus.

[0100] In another embodiment, the present invention includes a tissue culture system. In various aspects, the culture system comprises an ECM composition described herein, such as being contained in a two-dimensional or three-dimensional support material. In another aspect, the ECM composition described herein serves as a support for the growth of various cell types or as a two-dimensional or three-dimensional support. For example, the culture system can be used to support the growth of cells of the present invention. In one aspect, the culture system can be used to support the differentiation of cells.

[0101] ECM is known to be secreted by certain cells and is mainly composed of fibrous proteins, polysaccharides, and other trace components. Its components include structural elements, such as collagen and elastin; adhesion proteins, such as glycoproteins fibronectin, laminin, vitronectin, thrombospondin I, and tenascin; proteoglycans, such as decorin, biglycan, chondroitin sulfate, and heparin sulfate; and glycosaminoglycans (GAGs), such as hyaluronic acid (HA).

[0102] In one embodiment, the ECM composition may include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen type I, III, and IV), glycosaminoglycans, cytoplasm, reticular fibers, and thrombospongin. Preferably, the human ECM is used to culture endoderm of an embryo. In one embodiment, the human ECM comprises collagen, laminin, fibronectin, tenascin, elastin, and several proteoglycans and glycosaminoglycans.

[0103] In another embodiment, it is preferable to culture cells on a solid support containing a reconstituted basement membrane, where the membrane can be obtained by extracting and preparing from a suitable cell tissue that is contained in a thin, membrane-forming extracellular matrix present beneath the cell layer in vivo and contains proteins and glycoproteins, such as laminin, collagen type IV and heparin sulfate proteoglycans, as well as various cell growth factors and activators.

[0104] In one embodiment, the important primary extracellular matrix component is fibrous collagen, particularly collagen type I. However, other fibrous and non-fibrous collagens include collagen types II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, and so on.

[0105] The ECM compositions of the present invention can be processed in various ways. Therefore, in one embodiment, the present invention includes a tissue culture system. In various aspects, the culture system consists of the ECM compositions described herein. The ECM compositions of the present invention can be incorporated into a tissue culture system in various ways. For example, the compositions can be incorporated as a coating by impregnating a three-dimensional scaffold material described herein, or as an additive to a culture medium for culturing cells. Therefore, in one aspect, the culture system may include a three-dimensional support material impregnated with any of the ECM compositions described herein, such as growth factors or embryonic proteins.

[0106] While MSCs and their associated cell matrix can ultimately reach a fully differentiated state, which may be desirable in some situations (for example, when cells are used to replicate a histologically mature, complete tissue), it is not necessary for all administered cells to be fully differentiated for a successful procedure. The MSCs in the cell matrix only need to differentiate to a point sufficient to treat a mammal. This point may be reached before or after administering the matrix to the patient.

[0107] Differentiation occurs when the cells in the matrix express essentially the same phenotype as the mature cells at the implantation site. For example, for the purposes of defining this invention, MSCs in a cell matrix implanted in the lung are differentiated when they express essentially the same proteins as those expressed by the lung, for example, alveolar epithelial cells. Antibodies against lung markers are commercially available or readily obtainable.

[0108] Differentiated cells can also be identified by their macroscopic morphology and by the connections they form with other cells. For example, cells differentiating into lung cells can develop complex morphologies resembling bronchioles. For instance, the present invention is based on a novel discovery that various MSC types (e.g., bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs)) undergo significant changes in lung epithelial marker expression depending on the culture conditions, including the substrate or scaffold on which they are cultured. For example, BM-MSCs differentiate into cell types and tissues associated with type II alveolar epithelial cells, while AT-MSCs differentiate into cell types and tissues associated with Clara cells.

[0109] The number of cells introduced into and onto a decellularized organ to create an organ or tissue depends on the organ (e.g., organ type, organ size, and weight) or tissue, as well as the type and developmental stage of the regenerative cells. Different types of cells may have different tendencies with respect to the population density they will reach. Similarly, different organs or tissues may be cellularized at different densities. For example, a decellularized organ or tissue may be seeded with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) regenerative cells, or a decellularized organ or tissue may have attached thereto about 1,000 cells / mg of tissue (wet weight, i.e., before decellularization) to about 10,000,000 cells / mg of tissue (wet weight).

[0110] Cells can be introduced into a decellularized organ or tissue by injection into one or more locations. Furthermore, two or more types of cells (i.e., a cell cocktail) can be introduced into a decellularized organ or tissue. For example, a cell cocktail can be injected into multiple locations within the decellularized organ or tissue, or different cell types can be injected into different parts of the decellularized organ or tissue. Instead of injection, or in addition to injection, regenerative cells or cell cocktails can be introduced by perfusion into the decellularized organ or tissue via cannula insertion. For example, cells can be perfused into the decellularized organ using perfusion medium, which can then be replaced with expansion medium and / or differentiation medium to induce the growth and / or differentiation of regenerative cells. In the case of lung tissue, cells can be introduced into the airway compartment through the trachea, into the vascular compartment through the pulmonary artery or pulmonary vein, or both.

[0111] During recellularization, the organ or tissue is maintained under conditions that allow at least a portion of the regenerative cells to multiply and / or differentiate within and on the decellularized organ or tissue. These conditions include, but are not limited to, appropriate temperature and / or pressure, electrical and / or mechanical activity, force, appropriate amounts of O2 and / or CO2, appropriate amounts of humidity, and sterile or near-sterile conditions. During recellularization, the decellularized organ or tissue and the cells attached thereto are maintained in a suitable environment. For example, cells may require nutrient supply (e.g., nutrients and / or carbon sources such as glucose), exogenous hormones or growth factors, and / or a specific pH.

[0112] The cells may be allogeneic to the decellularized organ or tissue (e.g., a human decellularized organ or tissue seeded with human cells), or the regenerative cells may be heterogeneous to the decellularized organ or tissue (e.g., a pig decellularized organ or tissue seeded with human cells).

[0113] In some cases, organs or tissues produced by the methods described herein are transplanted into a patient. In such cases, the cells used to recellularize the decellularized organ or tissue can be obtained from the patient so that the regenerated cells become autologous cells to the patient. The patient's cells can be obtained, for example, from blood, bone marrow, tissue, or organs at different stages of life (e.g., in the neonatal period, prenatal or perinatal period, during adolescence, or as an adult) using methods known in the art. Alternatively, the cells used to recellularize the decellularized organ or tissue may be allogeneic cells to the patient (i.e., obtained from one of identical twins), or the cells may be human lymphocyte antigen (HLA) matched cells obtained, for example, from a relative of the patient or from an HLA-matched individual unrelated to the patient, or the cells may be allogeneic cells to the patient obtained, for example, from a non-HLA-matched donor.

[0114] Regardless of the cell source (e.g., whether autologous or not), decellularized solid organs can be autologous, allogeneic, or heterogeneous for the patient.

[0115] In certain cases, decellularized tissue may be recellularized in vivo by cells (e.g., after the tissue has been transplanted into an organism). In vivo recellularization may be carried out as described above, for example, using any of the cells described herein (e.g., injection and / or perfusion). Alternatively, in vivo seeding of decellularized organs or tissues by endogenous cells may occur spontaneously or be mediated by factors delivered to the recellularized tissue.

[0116] treatment This invention provides compositions and methods for treating various lung diseases and conditions using the cells of the present invention. In some examples, the cells included genetically modified cells.

[0117] The present invention may similarly include treating one or more lung diseases or conditions by administering the cells of the present invention. In a preferred embodiment of the present invention, the composition may be administered intratracheally. In the most preferred embodiment of the present invention, intratracheal administration involves contacting or exposing lung tissue, e.g., alveoli, to the cells of the present invention.

[0118] The compositions and methods of the present invention may be used to treat or suppress any lung disease or condition in which it may be desirable to contact one or more types of lung tissue with the composition comprising the cells of the present invention. As used herein, disease or condition means any disease or condition that results in pathological changes in the function or structure of the lung. Exemplary diseases or conditions include, but are not limited to, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), emphysema, cystic fibrosis (CF), pulmonary dysplasia, and pulmonary hypertension, and chronic obstructive pulmonary disease (COPD).

[0119] The compositions and methods of the present invention may be used to treat alveolar damage caused by any disease or condition. Exemplary diseases or conditions include, but are not limited to, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), emphysema, and chronic obstructive pulmonary disease (COPD).

[0120] The compositions and methods of the present invention may be used to treat or prevent any oxygen-induced lung injury, disease, or condition. Exemplary diseases or conditions include, but are not limited to, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), emphysema, cystic fibrosis (CF), pulmonary dysplasia, pulmonary hypertension, and chronic obstructive pulmonary disease (COPD).

[0121] In relation to gene therapy, cells can be treated with the gene of interest prior to delivery to the recipient. In some cases, such cell-based gene delivery can demonstrate significant advantages over other gene delivery methods to the lung, such as inhalation of adenovirus gene delivery vectors. This advantage of cell-based gene delivery to the host stems from the understanding that inhaled gene vectors typically result in low cell transduction efficiency due to barriers imposed by the mucus layer and the host immune system. Delivery of therapeutic genes pre-inserted into cells avoids the problems associated with the entry of gene therapy vectors into recipient lung cells.

[0122] Accordingly, the present invention provides the use of genetically modified cells cultured by the method of the present invention. Genetic modification may result in, for example, the expression of an exogenous gene ("transgene") or a change in the expression of an endogenous gene. Such genetic modification may have therapeutic benefits. Alternatively, genetic modification may also serve as a means for tracking or identifying such modified cells after, for example, implanting the composition of the present invention into an individual. Cell tracking may include tracking the migration, assimilation, and survival of the transplanted genetically modified cells. Genetic modification may also include at least a second gene. The second gene may encode, for example, a selectable antibiotic resistance gene or other selectable marker.

[0123] Proteins useful for tracking cells include, but are not limited to, green fluorescent protein (GFP), any of the other fluorescent proteins (e.g., enhanced green, cyan, yellow, blue, and red fluorescent proteins; Clontech, Palo Alto, CA), or other tag proteins (e.g., LacZ, FLAG-tag, Myc, His6, etc.).

[0124] When the purpose of genetically modifying cells is to produce biologically active substances, these substances will generally be useful for treating a given disorder. For example, it may be desirable to genetically modify cells so that they secrete specific growth factor products associated with bone or soft tissue formation. Growth factor products that induce the growth of other endogenous cell types involved in tissue repair are also useful. For instance, growth factors that stimulate endogenous capillary and / or microvascular endothelial cells may be helpful in repairing soft tissue defects, especially large-volume defects.

[0125] The cells of the present invention can be genetically modified by introducing exogenous genetic material into the cells to produce molecules beneficial to cell culture, such as trophic factors, growth factors, and cytokines. Furthermore, by genetically modifying cells to produce such molecules, the cells can provide additional therapeutic effects to mammals when transplanted into those that require them. For example, genetically modified cells can secrete molecules beneficial to cells adjacent to the transplantation site in the mammal.

[0126] Lung cells can be genetically modified using any method known to those skilled in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and Ausubel et al., Eds, (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY). For example, lung cells are exposed to an expression vector containing nucleic acid with a transgene so that the nucleic acid is introduced into the cell under conditions suitable for the transgene to be expressed in the cell. A transgene is generally an expression cassette containing a polynucleotide functionally linked to a suitable promoter. The polynucleotide can encode a protein or a biologically active RNA (e.g., antisense RNA or ribozyme). Therefore, polynucleotides can encode genes that confer resistance to toxins, hormones (e.g., peptide growth hormone, hormone-releasing factors, sex hormones, adrenocorticotropic hormone, cytokines (e.g., interferons, interleukins, lymphokines, etc.)), cell surface-bound intracellular signaling molecules (e.g., cell adhesion molecules, hormone receptors, etc.), and factors that promote differentiation of a given lineage (e.g., bone morphogenetic proteins (BMPs)).

[0127] Within the expression cassette, coding polynucleotides are functionally ligated to appropriate promoters. Examples of appropriate promoters include prokaryotic and viral promoters (e.g., retroviral ITRs, LTRs, pre-early viral promoters (IEp), e.g., herpesvirus IEp (e.g., ICP4-IEp and ICP0-IEEp), cytomegalovirus (CMV) IEp, and other viral promoters, e.g., Roussarcoma virus (RSV) promoter and mouse leukemia virus (MLV) promoter). Other appropriate promoters include eukaryotic promoters, e.g., enhancers (e.g., rabbit β-globin regulators), constitutively active promoters (e.g., β-actin promoter), signal-specific promoters (e.g., inducible promoters such as the RU486-responsive promoter), and tissue-specific promoters. Selecting a promoter suitable for driving gene expression in relation to a predetermined cell is well within the capabilities of the art. An expression cassette may contain two or more coding polynucleotides and may include other elements as desired (e.g., polyadenylation sequences, sequences encoding membrane insertion signals or secretion readers, ribosome entry sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.)).

[0128] The expression cassette containing the transgene should be incorporated into a gene vector suitable for delivering the transgene to cells. Depending on the desired end use, any of these vectors can be used to genetically modify cells (e.g., plasmids, naked DNA, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, papillomaviruses, retroviruses, etc.). Any method for constructing the desired expression cassette within such a vector can be used, many of which are well known in the art (e.g., direct cloning, homologous recombination, etc.). The choice of vector will largely determine the generally known methods used to introduce the vector into cells (e.g., protoplast fusion, calcium phosphate precipitation, gene guns, electroporation, transfection with DEAE dextran or lipid carriers, infection with viral vectors, etc.).

[0129] Examples of techniques sufficient to enable those skilled in the art to perform in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), and other DNA or RNA polymerase-mediated techniques, can be found in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0130] Once a nucleic acid for a particular protein has been cloned, a person skilled in the art can express the recombinant gene in various lung cells. It is expected that a person skilled in the art will know of numerous expression systems that can be used to express the desired transgene.

[0131] lung cells Differentiation is non-limited Ca 2+Differentiation can be induced using one or more differentiation agents, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), transformation growth factor (TGF), cytokines such as interleukin, interferon, or tumor necrosis factor, retinoic acid, transferrin, hormones such as androgens, estrogens, insulin, prolactin, triiodothyronine, hydrocortisone, or dexamethasone, sodium butyrate, TPA, DMSO, NMF (N-methylformamide), DMF (dimethylformamide), or matrix elements such as collagen, laminin, or heparan sulfate.

[0132] In one embodiment, MSCs can be induced to differentiate into cells having a lung phenotype. For example, MSCs can be induced to differentiate into type II alveolar cells, which are also known as type II lung cells. In another embodiment, MSCs can be induced to differentiate into Clara cells.

[0133] A culture medium can be used that contains one or more of the following: pituitary gland extract (e.g., bovine pituitary gland extract), steroid hormones (e.g., hydrocortisone, or a salt thereof such as acetate), growth factors (e.g., epidermal growth factor, preferably human epidermal growth factor), catecholamines (e.g., epinephrine, either racemic or enantiomer), iron-binding proteins (e.g., transferrin), insulin, vitamins (e.g., retinoic acid), thyroid hormones (e.g., triiodothyronine), serum albumin (e.g., bovine or human serum albumin, including recombinant preparations), antibiotics (e.g., aminoglycoside antibiotics such as gentamicin), and / or antifungal agents (e.g., amphotericin-B). For example, the medium may contain hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, and bovine serum albumin, and in some embodiments may further contain retinoic acid, pituitary gland extract, and epinephrine. Cambrex's SAGM™ medium (catalog CC-3118) is particularly useful for differentiating MSCs into desired lung cells.

[0134] The inventors were able to obtain a pure population of lung cells through the use of appropriate differentiation factors and culture conditions. In one embodiment, the lung cells are peripheral lung cell types, preferably alveolar cells, more preferably type I or type II alveolar cells. In another embodiment, the lung cells are Clara cells.

[0135] In some embodiments, the method of the present invention efficiently induces the direct differentiation of MSCs into alveolar type II cells. In some embodiments, the method results in a substantially pure population of alveolar type II cells (e.g., at least 95% alveolar type II phenotype).

[0136] In some embodiments, the method of the present invention efficiently induces the direct differentiation of MSCs into Clara cells. In some embodiments, the method yields a substantially pure population of Clara cells (e.g., a phenotype of at least 95% Clara cells).

[0137] Differentiation into lung cells (e.g., alveolar type II cells) can be confirmed, for example, by lung morphology assessed by light microscopy, and by the presence of laminae and microvacuoles assessed by transmission electron microscopy. Laminae are secretory lysosomes that function as storage-type lung surfactant, i.e., surfactant protein C (SPC), an intrinsic membrane protein expressed only in alveolar type II cells. The presence of SPC mRNA can be detected by reverse transcriptase PCR, and the presence of SPC protein can be detected by immunofluorescence staining. Clara cell differentiation can be assessed by detecting the presence of CCSP. [Examples]

[0138] Experimental Examples The present invention will be described in more detail below with reference to experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples, but rather as encompassing all variations that become apparent as a result of the teachings provided herein.

[0139] Without further explanation, those skilled in the art will likely be able to create and utilize the compounds of the present invention and practice the claimed methods using the foregoing description and the following exemplary examples. The following examples, therefore, specifically point out preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure.

[0140] Example 1: Epithelial cell differentiation of human mesenchymal stromal cells changes depending on the original tissue when cultured in a decellularized pulmonary scaffold. In the experiments presented herein, the potential of human-derived BM-MSCs and hAT-MSCs to contribute to lung epithelium after culture on a rat decellularized lung matrix was evaluated. hBM-MSCs and hAT-MSCs were found to be able to adhere and grow after seeding onto lung scaffolds. After 7 days of culture on a decellularized rat lung scaffold in small airway growth medium (SAGM), hBM-MSCs expressed the type 2 lung cell marker pro-SPC at the RNA and protein levels, secreted surfactants into the medium, and contained laminae as indicated by transmission electron microscopy (TEM). Furthermore, hBM-MSC cells, after culture in a rat bioreactor, produced cells positive for the proximal airway marker cytokeratin-5. In contrast, hAT-MSCs produced not only Clara-like cells lining the airways at anatomically accurate locations, but also pro-SPC-positive cells. However, unlike hBM-MSCs, hAT-MSCs do not produce cytokeratin-5 positive cells after culturing in a lung bioreactor.

[0141] The effect of substrate matrix composition on mesenchymal stromal cell differentiation was also investigated by seeding hBM-MSCs and hAT-MSCs onto tissue culture dishes coated with human ECM, Matrigel, laminin, collagen 1, collagen 4, and fibronectin. It has been demonstrated herein that matrix surface coating affects the proportion of MSCs expressing lung epithelial markers during growth in standard tissue culture flasks. Furthermore, when hBM-MSCs were cultured on decellularized liver, it was found that MSCs seeded on liver slices did not retain the same set of epithelial markers they exhibited when cultured on lung matrix. These data suggest that the decellularized lung scaffold holds a "zip code" that directs cell differentiation. These data demonstrate that human-derived hBM-MSCs and hAT-MSCs can give rise to multiple lung epithelial cell types when placed on decellularized lung.

[0142] The materials and methods used in these studies will now be described.

[0143] Isolation and characterization of human bone marrow and adipose tissue mesenchymal stromal cells Fresh, untreated human bone marrow samples were obtained from Lonza, Allendale, NJ, USA (catalog number 1M-125). Three whole donor samples were obtained: two women and one man, aged 22–29 years. 1 cm of each sample was placed in high-glucose DMEM containing 10% FBS. 2 5 x 10 cells per unit 5 Bone marrow cells were plated at a density of 10 cells. The culture medium was changed every 2-3 days. Only cells with a low passage count (5 or less) were used in the experiment. Cells were passaged in a 1:3 ratio every 7-10 days. Cells were characterized by flow cytometry (BD LSR II) for the expression of CD90, CD105, CD73, and CD45 (all antibodies obtained from eBiosciences).

[0144] AT-MSCs were obtained from three donors aged 44-63 years. Aspirated adipose tissue was washed twice with DPBS, and then digested with 0.15% collagenase type 1 (Gibco, Grand Island, NY, USA, catalog number 17100-017) in DMEM at 37°C for 60 minutes. Digestion of the digested material was stopped by adding 10% FBS / DMEM, followed by centrifugation, resuspension in 10% FBS / DMEM, and filtration through a 100 μm filter. Nutrients were supplied to the cells every 2-3 days.

[0145] FACS Single cells were fixed in 2% paraformaldehyde solution for 10 minutes and washed twice in PBS for 5 minutes each. Cells were incubated with 10% FBS, 0.2% Triton X-100 containing the diluted antibody of interest. Cells were incubated on ice in the dark for 25 minutes with the following antibodies (all obtained from eBiosciences, San Diego, CA, USA): CD45-PE (12-9459-41), CD90-FITC (11-0909-41), CD73-PE (12-0739-41), and CD105-APC (17-1057-41). Further antibodies used were ProSPC 1 / 100 (Millipore, Billerica, MA, USA: ab 3786) and CCSP 1 / 100 (Millipore 07-623). The secondary antibodies for all of these antibodies were species-appropriate Invitrogen Alexa Fluors diluted to 1 / 500. Isotype controls used included anti-mouse IgG-PE (eBiosciences 12-4714), anti-mouse IgG-FITC (eBiosciences 11-4724), anti-mouse IgG APC (eBiosciences 17-4015-80), rabbit IgG-FITC (eBiosciences 11-4614-80), and purified rabbit IgG (Invitrogen 02-6102). In addition to the isotype controls, secondary-only antibody controls were run in parallel.

[0146] Decellularization and redissemination of rat lung samples Adult Sprague Dawley rat lungs (3-5 months old) were decellularized as previously described (Petersen et al., 2010, Science 329:538-541). Briefly, the rats were euthanized by IP injection of pentobarbital sodium (Euthasol). The rat lungs were resected, and cannulas were inserted into the trachea and pulmonary artery. A 10 ml mixture of heparin (50 U / ml) and sodium nitroprusside (1 μg / ml) was injected by gravity through the pulmonary artery. 500 ml of a pH 12 decellularization solution consisting of 8 mM CHAPS, 25 mM EDTA, and 1 M NaCl in PBS was injected into the pulmonary artery at a constant pressure of 20 mmHg at 37°C. Then, 10 ml of benzonase was injected into the lungs through the airway and incubated at 37°C for 1 hour. Cellular residues were washed away with 2.5 L of PBS. The lungs were incubated for at least 16 hours in a mixture of antibiotics and antifungals (1% gentamicin, 4 mg / ml amphotericin, 10% penicillin / streptomycin) before seeding the cells. During this incubation, the solution was perfused through the pulmonary artery at 1 mL / min. Before seeding the cells, the antibiotic / antifungal solution was removed from the lungs and replaced with PBS. The lungs were perfused with PBS for an additional 30 minutes before seeding the cells. 2.5 × 10⁶ cells were then fed into one decellularized right upper rat lung lobe via the trachea. 6 ~10×10 6 Cells were seeded as individual boluses. The cultures were maintained in SAGM (Lonza, Allendale, NJ; catalog number CC-3118) for 7 days while perfusing with medium through the pulmonary artery at 1 ml / min. The medium was changed every 2-3 days.

[0147] immunohistochemistry The lungs were injected with 10% formalin solution through the airway and left in 10% formalin at room temperature for 4 hours with constant shaking. The lungs were embedded in paraffin and sectioned into 5 micron sections. The sections were deparaffinized according to the standard rehydration alcohol / xylene series. Antigen recovery was performed by incubation of the rehydrated tissue sections in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween-20 pH 9.0) at 75°C for 20 minutes. The tissue sections were then cooled to room temperature for a further 20 minutes. Before immunostaining, the sections were washed once in PBS. The sections were incubated in blocking reagent (10% NGS or FBS in PBS, 0.2% Triton X-100) for 45 minutes. Primary antibodies (CCSP 1 / 50: Millipore catalog number 07-623; ProSPC 1 / 100: Millipore: # ab 3786; Caveolin-1 1 / 100: Abcam, Cambridge, UK # 39541; α-smooth muscle actin 1 / 100: Dako, Glostrup, Denmark catalog number M0851) were incubated at room temperature for 2 hours or overnight at 4°C. Sections were washed three times in PBS for 3 minutes each, and then incubated with secondary antibodies (all species-specific secondary antibodies from the Invitrogen Alexa Fluor series diluted to 1 / 500) at room temperature for 45 minutes. Tissue sections were mounted in Vector Labs Vectashield mounting medium containing DAPI (Vector cat., Olean, NY # H1200).

[0148] Processed sections were imaged using a Zeiss fluorescence microscope, and images were acquired using Volocity software. Confocal microscope images were acquired using a Leica TCS SP5.

[0149] Real-time quantitative RT-PCR Total RNA was extracted from cells using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized using the SuperScript First-Strand Synthesis System according to the manufacturer's protocol (Invitrogen), using random hexamers as primers. An equal mixture of the products was used as a template for PCR amplification. Reactions were carried out in a volume of 25 μl using iQ (trademark) SYBR Green Supermix (Bio-Rad, Hercules, NY) and 200 nM each of the forward and reverse primers indicated, using the iCyler and iQ software (Bio-Rad). Each sample was run in triplicate. PCR conditions included an initial denaturation step at 95°C for 4 minutes, followed by 40 cycles of PCR consisting of 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. The mean threshold cycle (Ct) value from triplicate PCR reactions for the gene of interest (GOI) was normalized to the mean GAPDH Ct value from the same cDNA sample. The fold change in GOI transcript levels between sample A and sample B was equal to 2 -ΔΔCt where ΔCt = Ct (GOI) - Ct (GAPDH) and ΔΔCt = ΔCt (A) - ΔCt (B) as described elsewhere in this specification. The primers used are described elsewhere in this specification.

[0150] statistical analysis All statistical analyses were performed using Origin software (OriginLab, Northampton, MA). Data were expressed as mean ± s.e.m. (standard error of measurement). A t-test was performed to evaluate whether the two groups were significantly different from each other, and p ≤ 0.05 was considered statistically significant.

[0151] TEM The modified protocol of Schmiedl et al. 2005 was followed (Schmiedl, et al., 2005, Histochem Cell Biol 124(6):465-76). Natural rat lungs and recellularized lungs were inflated and fixed at 37°C for 30 minutes with 2.5% glutaraldehyde / 2.0% paraformaldehyde in 0.2 M sodium cacodylate, followed by incubation at 4°C for 2 hours. The fixed tissues were washed with 0.1 M sodium cacodylate. The tissues were post-fixed in 1% OsO4 for 2 hours and then stained en block uranyl acetate. The tissues were dehydrated in a standard ethanol series and embedded in EPON. Sections (70 nm) were collected and post-stained with uranyl acetate and lead citrate. Images were obtained using a Philips Tecnai transmission electron microscope.

[0152] Coating of matrix proteins for cell culture hBM-MSCs and hAT-MSCs were cultured for 7 days on various extracellular proteins containing fibronectin (50 μg / ml), collagen I (100 μg / ml), collagen IV (50 μg / ml), Matrigel (1:80), and a mixture of human ECM proteins (1:100) (consisting of collagen, laminin, fibronectin, tenascin, elastin, and several proteoglycans and glycosaminoglycans; Sigma). Fibronectin, collagen I, collagen IV, and laminin are major components of the lung matrix.

[0153] SPC's Enzyme-linked Immunoassay (ELISA) To quantify secreted SPC (Life Science Advanced Technology) according to the manufacturer's instructions, ELISA was performed on cell media collected from the supernatant of hBM-MSCs and hAT-MSCs cultured on rat cell-free lung scaffolds. SPC values ​​were normalized to the total number of cells, and the values ​​of the experimental samples were subtracted from those in fresh SAGM medium only.

[0154] The results of the experiment will now be described.

[0155] Characterization of MSCs isolated from bone marrow and adipose tissue hBM-MSCs were obtained from fresh, isolated bone marrow samples (Figures 1A-1C). Unfractionated bone marrow was placed on tissue culture flasks, and the resulting adherent cells were immunophenotyped for the expression of standard MSC clusters of differentiation markers. Early-passage hBM-MSCs (passages 2-4) were over 93% positive for CD90, CD105, and CD73, but the majority were negative for CD45 (Figure 1B). Previous studies had shown that subfractions of cultured MSC-like cells could express epithelial markers (Wong, et al., 2009, J Clin Invest 119(2):336-48), so cells were also assayed for the expression of epithelial markers including CCSP, pro-SPC, and cytokeratin 5 (Figure 1C). FACS analysis revealed that hBM-MSCs expressed epithelial markers after standard culture in 10% FBS / DMEM. The data presented herein suggest that hBM-MSCs are positive for CCSP (80%), pro-SPC (63%), and cytokeratin-5 (77%) by FACS analysis (Figure 1C). As a negative control for these experiments, unfractionated whole bone marrow mononuclear cells were analyzed by FACS and were uniformly negative for evaluation of lung epithelial markers (Figure 12).

[0156] hAT-MSCs were isolated from freshly harvested aspirated adipose tissue (Figures 1D-1F). Cells were isolated by collagenase-1 tissue digestion and placed on tissue culture flasks. These cells were maintained in 10% FBS / DMEM medium for expansion. Immunophenotyping of hAT-MSCs confirmed their identity as CD90+ / CD105+ / CD73+ and CD45- (Figure 1E). Similar to hBM-MSCs, hAT-MSCs were immunopositive for various epithelial markers by FACS (Figure 1F). Interestingly, there was a difference in the total number of cells expressing epithelial markers between hAT-MSCs and hBM-MSCs. In contrast to the hBM-MSC population, approximately half of the hAT-MSC population was CCSP-positive (44%); hAT-MSCs were also pro-SPC-positive (51%) and cytokeratin 5-positive (91%) (Figure 1F).

[0157] Regeneration of rat cell-free matrix by MSCs in a lung bioreactor hBM-MSCs re-seeded on rat lung cell-free matrix To understand whether adipose- or bone marrow-derived MSCs can recellularize the rat cell-free matrix and whether these cells can exhibit an epithelial phenotype after placement on the cell-free matrix, MSCs were cultured in the biomimetic rat lung bioreactor system described above (Petersen et al., 2010, Science 329:538-541). Before seeding the MSCs onto the decellularized lung scaffold, H&E histological features and DAPI staining of the native and decellularized lung matrices were observed. These analyses confirmed that there were no residual native lung cells in the decellularized lung (Figure 2A-B).

[0158] hBM-MSCs cultured for 2-4 passages were seeded through the trachea into one right upper lung lobe of a decellularized rat lung at a density of 2.5-10 million cells. These cells were injected as a bolus into the trachea and cultured in small airway growth medium (SAGM) in a lung bioreactor for 7 days. This resulted in cells with uniformly fibroblast-like morphology, and after 1 week of culture, almost all cells expressed α-sma, a marker for myofibroblasts. Following a pilot experiment using 10% FBS / DMEM, SAGM was selected as a potentially suitable medium for culturing recellularized lung cells (Figure 7). SAGM was selected as a good candidate for promoting lung epithelial differentiation because its retinoic acid and human epidermal growth factor had been shown to promote the proliferation and epithelial differentiation of pluripotent cells (Rackley & Stripp, 2012, J Clin Invest 122:2724-2730; Lenssen & Stolk, 2007 Int J Chron Obstruct Pulmon Dis. 2(2): 131-139). In vitro pilot experiments were conducted in which MSCs were grown in tissue culture flasks with SAGM medium or in 10% FBS / DMEM. These experiments demonstrated that MSCs grown in SAGM lacked α-sma-positive cells, but the cells maintained CCSP expression at similar levels (Figures 7C-7F).

[0159] In vitro experiments showed that SAGM was used to suppress the amount of cells expressing α-sma and thus promoting lung epithelial differentiation. However, before seeding hBM-MSCs into cell-free lungs, the cells were maintained in 10% FBS / DMEM medium to promote robust proliferation.

[0160] H&E staining of redisseminated hBM-MSC lung cells cultured in SAGM for 7 days demonstrated a more cuboidal appearance in the adhered cells compared to cells grown in 10% FBS / DMEM in a lung bioreactor (Figures 2C and 7). Consistent with in vitro culture, α-sma immunostaining was almost completely absent in the recellularized hBM-MSC rodent lungs. Additional staining for lung epithelial markers confirmed that 65–70% of the adhered cells expressed the type 2 lung cell marker pro-SPC (Figure 2D). Cytokeratin-5, a marker expressed by basal epithelial cells of the airways, was also present in some of the adhered cells (Figure 2F). Interestingly, despite the initial hBM-MSC population expressing CCSP by immunostaining and FACS, no CCSP-positive cells were present after culture in the lung bioreactor (Figure 2E; Figure 1C). Furthermore, the attached cells were negative for P63, a marker for basal cells, and caveolin-1, a marker for type 1 lung cells.

[0161] To gain complete insight into the function and sufficient differentiation state of pro-SPC-positive cells derived from hBM-MSCs, we attempted to identify not only surfactant vesicles secreted by these cells but also laminae in type 2 lung cells using transmission electron microscopy (Figure 2H). Identification of laminae by transmission electron microscopy in native lung type 2 cells is a method often used for definitive identification of type 2 cells (Schmiedl, et al., 2005, Histochem Cell Biol 124(6):465-76). The presence of laminae can also indicate the function of these cells (Kassmer & Krause, 2010, Experimental Hematology 38:564-573). Laminae act as storage sites for secreted surfactants and lipids contained within type 2 cells. Both human and rat type 2 lung cells are characterized by the presence of laminae. As a positive control, we also investigated the presence of laminae within type II lung cells in native rat lung (Figure 2B, blue arrows). These laminae are approximately 500 nm long and are characterized by high-electron-density deposits within structures formed from concentric vortices. Furthermore, another characteristic feature of native type II lung cells is the presence of secretory vesicles (Figure 2G, white arrows).

[0162] Decellularized lung scaffolds cultured for 7 days, with only hBM-MSCs seeded and other cell types removed, were also assayed for the presence of laminae in addition to secretory ducts in adherent cells (Figure 2H). These hBM-MSC-derived cells possessed abundant, electron-dense laminae and numerous secretory vesicles (arrows and chevron symbols in Figure 2H). Morphologically, native rat type II lung cells were very similar to hBM-MSCs cultured for 7 days in a bioreactor on the lung matrix in the presence of SAGM. In summary, these data suggest that hBM-MSCs not only express type II lung cell-related markers at the protein level but also possess function-related cytoplasmic structures similar to those found in native type II cells. Therefore, these data demonstrate that hBM-MSCs can exhibit the phenotype of type II alveolar epithelial cells when expanded and cultured under appropriate conditions.

[0163] hAT-MSCs re-seeded on rat lung cell-free matrix To determine the ability of hAT-MSCs to engraft on decellularized lung tissue, cells 2.5-10 × 10 6 The cells were seeded as a bolus into decellularized rodent lung lobes (upper right) and cultured in SAGM medium for 7 days under the same conditions as hBM-MSCs. From H&E tissue slides of the seeded lungs, it was demonstrated that hAT-MSCs, unlike hBM-MSCs which can adhere to the entire matrix but not to the airways, possess a specific affinity for adhering to and repositioning in the airways (Figure 3A-B). Immunostaining revealed that cells adhering to the airways were positive for the Clara cell marker CCSP. This was in contrast to hBM-MSCs. hBM-MSCs did not maintain CCSP expression after culture on the lung matrix, and these cells did not adhere to the airways.

[0164] Furthermore, hAT-MSCs cultured in a lung bioreactor were pro-SPC positive, a type 2 lung cell marker. Immunofluorescence against pro-SPC revealed cells with clear, punctate cytoplasmic staining for this marker (inset in Figure 3C). Another difference between cultured hAT-MSCs and hBM-MSCs was that hAT-MSCs did not produce cells positive for cytokeratin-5 by immunofluorescence, while hBM-MSCs did (Figures 3D and 2H). These data suggest that there are essential differences in the ability of MSCs derived from different tissue sources to rearrange themselves in the lung cell-free matrix.

[0165] Alveolar epithelial gene expression after lung bioreactor culture Gene expression of various peripheral lung epithelial markers was also evaluated in recellularized lung cells from hBM-MSCs and hAT-MSCs. Real-time qRT-PCR was used to assay the expression of surfactant protein C (type II), caveolin-1, and aquaporin 5 (AQP5, type I) (Figure 4).

[0166] Surfactant protein C (SPC) is widely used as a marker for type II lung cells and early lung progenitor cells. RT-PCR revealed increased SPC expression in hBM-MSC recellularized lung cells when bioreactor cultures at day 3 and day 7 were compared with MSCs grown in flasks (Figure 4A). These results indicate a gradual increase in the amount of SPC transcript present at day 3 and day 7, by 23-fold and 93-fold, respectively (Figure 4A).

[0167] Similar patterns of SPC expression were observed in lung scaffolds seeded with hAT-MSCs. Compared to hAT-MSCs grown in flasks, SPC expression increased from day 3 (36×) to day 7 (137×). The increase in SPC gene expression was even greater in lungs recellated from hAT-MSCs compared to lungs recellated from hBM-MSCs.

[0168] Furthermore, the expression of two specific alveolar type I markers, including caveolin-1 and AQP5, was evaluated in rat lung scaffolds seeded with hBM-MSCs and hAT-MSCs. Gene expression of both caveolin-1 and AQP5 increased over time in lung bioreactor cultures compared to MSCs grown on tissue culture flasks, when evaluated on days 3 and 7 (Figures 4B-F). While gene expression for these two type I cell markers increased over time in culture, protein expression of the type I markers caveolin-1 or aquaporin 5 based on immunostaining could not be detected.

[0169] Overall, qRT-PCR revealed that gene expression levels for SPC, caveolin-1, and AQP5 increased over time in bioreactors seeded on pulmonary scaffolds in both hBM-MSCs and hAT-MSCs. However, protein expression indicated by immunohistochemistry suggests that only pro-SPC among these genes is detectable in hBM-MSCs and AT-MSCs after 7 days on pulmonary scaffolds, while expression for type 1 markers is undetectable. While we do not wish to be bound by any particular theory, this apparent discrepancy is likely explained by protein levels that, depending on the sensitivity of qRT-PCR, are acceptable for detecting small amounts of mRNA expression from cells seeded on pulmonary scaffolds, but are not strong enough for a signal detectable by immunohistochemistry.

[0170] Functionality of type 2 lung cells derived from hBM-MSCs and hAT-MSCs To determine whether MSCs seeded on a decellularized lung scaffold provided functional improvement to the organ, we evaluated whether active surfactant secretion into the culture medium was observed by cells growing on the decellularized scaffold (Figure 5). hBM-MSCs were seeded into a decellularized lung lobe, 2 × 10⁶ cells, in the upper right. 6Lung lobes were seeded at a density of 100 cells and allowed to adhere to the matrix for 2 hours. After this, the lobes were cut in half, and the fragments were placed in a 6-well culture plate containing SAGM. The lung slices thus seeded were cultured for 3 or 7 days, and the culture medium was collected at these points. RT-PCR showed increased SPC gene expression between days 3 and 7 compared to decellularized scaffolds alone (Figure 5A, 5B). Bright-field microscopy of the active cultures revealed a visible layer of oily droplets that were largely concentrated near the lung fragments (Figure 5D, 5E). As a control to these experiments, scaffolds without seeded MSCs were placed under the same culture conditions. These controls did not contain oily droplets (Figure 8). Since the visible droplets may have been surfactant actively produced by the seeded cells, ELISA was performed to test for the presence of surfactant protein C (Figure 5C). Surfactant protein C was present in hBM-MSC slice cultures at 3.3 ng / mL and 0.26 / mL, respectively, on both days 3 and 7 (Figure 5C). Lung cultures seeded with hAT-MSCs contained even higher levels of surfactant protein present in the culture medium. Normalized SAGM from these cultures contained 5.55 ng / mL and 1.16 ng / mL, respectively (Figure 5C). These data suggest that seeded MSCs function similarly to type 2 lung cells in that they actively produce and secrete surfactant when cultured on a decellularized lung scaffold.

[0171] Influence of substrate matrix on MSC differentiation To further understand the effect of culture substrates on MSC differentiation, either hAT-MSCs or hBM-MSCs were cultured on tissue culture flasks coated with various extracellular matrix (ECM) proteins, including collagen 1, collagen IV, laminin, fibronectin, human ECM, and Matrigel (Figure 6). Collagen 1, collagen IV, laminin, and fibronectin were selected because they are representative components of the lung ECM, respectively (Petersen et al., 2012, Cells Tissues Organs 195:222-231; Cortiella et al., 2010, Tissue Eng Part A 16:2565-2580). Human ECM and Matrigel coating were selected to provide the cells with a mixed ECM composition. After a 7-day culture period in SAGM, the cell populations were characterized for epithelial marker expression by FACS and RT-PCR.

[0172] Data from FACS analysis experiments on hBM-MSCs cultured on variant substrates revealed significant differences in the cell populations expressing cytokeratin 5, CCSP, and pro-SPC depending on the ECM component on which the cells were cultured (Figures 6A, 9, and 10). Cells grown on collagen type 1 substrates had the lowest levels of cytokeratin 5 positivity; approximately 3.0% of the population was positive, which was about three times lower compared to cells grown on fibronectin-coated plastic flasks. Furthermore, the amount of CCSP-positive cells in the hBM-MSC population varied dramatically depending on the substrate on which they were grown. The largest CCSP-positive cell population was found in fibronectin-coated flasks (44%), while the lowest levels of CCSP-positive cells were observed in hBM-MSCs grown on collagen type 4 (27%). Similarly, the population of hBM-MSCs expressing pro-SPC differed among surface coatings, with the highest number of positive cells present in flasks coated with human ECM (54%), while the lowest number were found in cells grown on Matrigel (34%).

[0173] Furthermore, the behavior of hAT-MSCs grown on different ECM matrices differed in populations expressing CCSP, pro-SPC, and cytokeratin-5. For example, the pro-SPC-positive cell population ranged from 81% under human ECM conditions to 32% under collagen 1 conditions. The CCSP-positive cell population was also highest (10%) in flasks coated with human ECM compared to other surface coatings. While there were differences in the amount of cells expressing epithelial markers between hAT-MSCs and hBM-MSCs, phenotypic differences were also evident between populations, particularly those cultured on human ECM (Figures 6E, 6F). hAT-MSCs cultured on human ECM formed a lattice-like network across the culture dish, while hBM-MSCs maintained standard MSC morphology.

[0174] RT-PCR was performed as a further means to quantify RNA changes resulting from culture on different ECM substrates. Similar patterns of CCSP, cytokeratin-5, and SPC gene expression were observed by qPCR in hBM-MSCs and hAT-MSCs cultured on different ECM proteins, as seen by flow cytometry (Figure 6C, 6D). However, qPCR data showed that mixed human ECM proteins resulted in significantly higher SPC gene expression, lower CCSP, and lower cytokeratin-5 gene expression in both hBM-MSCs and hAT-MSCs at day 7 compared to cells cultured on other ECM proteins.

[0175] Overall, both hAT-MSC and hBM-MSC populations exhibit significant changes in lung epithelial marker expression depending on the culture substrate. Furthermore, these data suggest that the substrate alone can influence lung epithelial marker expression by mesenchymal stromal cells. These data provide insights into the differences in epithelial marker expression when comparing hAT-MSC and hBM-MSC cultured in a lung bioreactor.

[0176] Finally, the phenotype of hBM-MSCs seeded on decellularized liver matrix was analyzed in comparison to the phenotype of hBM-MSCs after lung bioreactor culture (Figure 11). hBM-MSCs were seeded on decellularized liver slices and cultured for 3 days, followed by immunohistochemical staining and H&E histological analysis. These data show that hBM-MSCs cultured on liver slices in either 10% FBS / DMEM or SAGM did not express cytokeratin 5 or CCSP, and only very rarely expressed pro-SPC, as confirmed by immunohistochemical staining (Figure 11). The culture period was limited to 3 days because longer periods result in lower MSC viability. These data further suggest that the ECM remaining after lung decellularization is involved in the ability of MSCs to differentiate and maintain the expression of lung epithelial markers.

[0177] Adipose and bone marrow-derived MSCs can adopt the phenotype of type II cells and become functional after being cultured in a decellularized rat lung bioreactor system. Previous research has led to the development of methods for successfully decellularizing rat lungs (Petersen et al., 2010, Science 329:538-541; Petersen et al., 2012, Cells Tissues Organs 195:222-231). These findings could ultimately make it possible to use decellularized human lung as a scaffold from which suitable donor cells can proliferate and differentiate into lung epithelial cell types (Badylak et al., 2011, Annu Rev Biomed Eng 13:27-53). Essential characteristics of the donor cell type include: 1) the ability to differentiate into lung epithelium or other cell types; 2) high proliferative capacity (these cells must be able to grow to a large number before being seeded onto the decellularized lung scaffold); 3) readily available from the patient (i.e., an autologous cell source, either differentiated cells or stem cells); and 4) lack of immunogenicity or induction of an immune response. Overall, these characteristics suggest the potential for using mesenchymal stromal cells as a source for re-seeding in decellularized lungs. While it has been previously shown that hBM-MSCs give rise to various epithelial cell types, including lung epithelium, some of these findings are still controversial (Krause et al., 2001, Cell 105: 1-9; Wong et al., 2009, Cytotherapy 11:676-687; Ortiz et al., 2003, PNAS 100:8408-8411; Wong et al., 2007, Am J Physiol Lung Cell Mol Physiol 293:L740-L752; Wang et al., 2006, Stem Cells 24:482-493). Furthermore, MSCs are less immunogenic than most cell types, do not express MHC class 2 markers, and do not elicit a potent immune response, as evidenced by the lack of T cell activation (Rasmusson et al., 2003, Transplantation, 76: 1208-1213).This study of human bone marrow and adipose tissue-derived MSCs in rat lung matrix points to clear and undeniable evidence of epithelial cell differentiation into at least type II alveolar cell phenotypes, and possibly other phenotypes as well.

[0178] The data presented herein demonstrate that human BM-MSCs, when placed on a decellularized rat lung matrix and cultured in SAGM, can express the type 2 lung cell marker pro-SPC and the proximal airway marker cytokeratin-5. Previous studies have failed to demonstrate any substantial contribution from BM-MSCs seeded on a decellularized lung scaffold to lung epithelium (Daly et al, 2012, Tissue Eng Part A 18: 1-16; Bonvillain et al., 2012, Tissue Eng Part A 18(23-24):2437-52). While we do not wish to be bound by any particular theory, this difference between our data and those of other studies can be partially explained by differences in the lung matrix decellularization process and the use of continuous perfusion of culture medium via the pulmonary artery in the bioreactor culture used in this study. In contrast, these other groups' studies used lung slice culture systems and lung scaffolds that likely retained different ECM components. Furthermore, while the decellularization method used herein utilizes 8 mM CHAPS, other groups have used 0.1% Triton-X in their decellularization protocols. Previous reports comparing CHAPS and SDS surfactants for decellularization have found differences in collagen and elastin retention after lung decellularization (Petersen et al., 2012, Cells Tissues Organs 195:222-231). Different matrix components remain after lung decellularization with CHAPS compared to decellularization with Triton-X, and consequently, there is a high possibility that this has an effect on the differentiation of seeded BM-MSCs. Another possibility, though less likely given the discrepancies between reports, is that this study used human-derived MSCs seeded on rat lungs, while Daly and his colleagues used mouse lungs and cells, and Bonvillain et al. used both macaque lungs and cells.

[0179] It is also shown herein that hAT-MSCs produce Clara-like cells that line the airways and express the CCSP protein, a feature not seen in any of the three human bone marrow donor samples assayed. Furthermore, like hBM-MSCs, hAT-MSCs also produce type 2-like cells, but not cells that are cytokeratin-5 positive after lung recellularization. These differences are particularly interesting considering that both MSC sources begin with common CD marker expression. However, as shown herein, the MSC population varies in relation to the population expressing the epithelial markers CCSP, pro-SPC, and cytokeratin-5. These differences in initial epithelial marker expression can lead to downstream variability among MSC sources after culture in lung bioreactors. Various reports have demonstrated differences in the differentiation potential of MSCs depending on the primary tissue (Vidal et al., 2008, Veterinary Surgery 37:713-724; Baer & Geiger, 2012, Stem Cells International 2012, 1-11; Al-Nbaheen et al., 2013, Stem Cell Rev 9(1):32-43; Hoffman et al., 2011, Stem Cells and Development 20: 1779-1792; Pevsner-Fischer et al., 2011, Stem Cell Rev and Rep 7:560-568). A comparison of gene expression among MSCs derived from bone marrow, adipose tissue, and skin revealed significant differences in their ability to express genes related to osteogenic and adipogenic lineages (Al-Nbaheen et al., 2013, Stem Cell Rev 9(1):32-43). Considering these results when comparing hAT-MSC sources and hBM-MSC sources, while we do not wish to be constrained by any particular theory, it is highly likely that further MSC sources may also possess different differentiation potential when cultured under appropriate lung biomimetic conditions.Of particular interest to evaluate are cells isolated from bronchoalveolar lavage fluid, so-called "pulmonary MSCs" that adhere to plastic and express standard markers associated with bone marrow MSCs (Hoffman et al., 2011, Stem Cells and Development 20: 1779-1792; Lama et al., 2007, J Clin Invest 117:989-996; Jarvinen et al., 2008, J Immunol. 181(6):4389-96).

[0180] Previous studies have demonstrated the ability of BM-MSCs to differentiate into lung epithelium, but these findings are not without debate (Kassmer & Krause, 2010, Experimental Hematology 38:564-573). These debates seem to stem mainly from the variability in experimental methods used by researchers, particularly the use of eGFP as a means of lineage tracking of cells of interest (Krause, 2008, Proc Am Thorac Soc 5:323-327). Our results further solidify the ability of hBM-MSCs to contribute to lung epithelium, as the starting material is decellularized lung and hBM-MSCs or hAT-MSCs. The decellularized lung in this study is completely devoid of lung epithelial cells (Figure 2B). This eliminates the possibility of misinterpreting contaminating cells from sources other than bone marrow or adipose tissue as lung epithelium.

[0181] Furthermore, we attempted to demonstrate that recellularized lung tissue exhibits functional improvements compared to cell-free lung scaffolds. One of the main functions of alveolar type II cells is the production of surfactant protein. Both hAT-MSC and hBM-MSC populations expressed surfactant protein C by immunofluorescence and RT-PCR and contained laminae as indicated by TEM. However, MSCs cultured in recellularized lung tissue actively produced SPC by ELISA and also contained visible surfactant droplets in the culture medium in contact with lung slices. The data presented herein suggest that hAT-MSCs can secrete up to 5.5 ng / mL of SPC into the culture medium at day 3 of culture, compared to 3.3 ng / mL secreted by hBM-MSCs. These data further support the idea that MSCs can adopt the phenotype of type II cells, but can also produce surfactant while adhering to a decellularized lung matrix. Interestingly, supporting the importance of culture medium in cell differentiation, there was no visible evidence of surfactant secretion from any MSC population under culture conditions maintained in 10% FBS (Figures 5 and 8).

[0182] To assess the importance of the epithelial cell layer (ECM) in regulating cell differentiation, MSCs were cultured in SAGM and different types of ECM, and gene expression levels for several epithelial markers were evaluated by RT-PCR, while population marker expression was assessed by FACS analysis. These data demonstrate that MSC populations differ significantly in terms of marker expression when grown on different ECM substrates. In related experiments, hBM-MSCs were cultured for 3 days on decellularized liver in either SAGM or 10% FBS (Figure 11). These experiments aimed to answer particularly fundamental, yet surprisingly, often under-evaluated, questions regarding the importance and influence of retained ECM on cell differentiation after culture on decellularized organs. These experiments showed that hBM-MSCs did not express cytokeratin-5 after culture on cell-free liver, and were largely negative for pro-SPC expression.

[0183] In summary, these experiments demonstrate that while MSCs derived from both adipose tissue and bone marrow can adopt the phenotype and function of type II cells after culture in a decellularized rat lung bioreactor system, only hAT-MSCs can colonize CCSP-positive cells in the airways. These data are important because various MSC sources may be utilized as donor cells for whole-organ regeneration in future lung recellularization efforts. For this purpose, the potential of alternative MSC sources, including those derived from umbilical cord and lung tissue, is being explored in either isolated or co-culture scenarios to enhance the recellularization process.

[0184] Any patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed in specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0185] Sequence information SEQUENCE LISTING <110> Yale Plaza <120> EPITHELIAL CELL DIFFERENTIATION OF HUMAN MESENCHYMAL STROMAL CELLS <150> US 61 / 866,570 <151> 2013-08-16 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 1 ccttcttatc gtggtggtgg t 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 2 tctccgtgtg tttctggctc at 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 actgggtttt ctgggtaggg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 4 atggtcttct tccgctcttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 ctacaagccc aacaacaagg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 6 catcgttgag gtgtttaggg t 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 7 gacaacagcc tcaagatcat cag 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 8 atggcatgga ctgtggtcat gag 23

Claims

1. A population of lung epithelial cells differentiated from mesenchymal stem cells (MSCs) by a method of differentiating mesenchymal stem cells (MSCs) into lung cells, At least 95% of lung epithelial cells express cytokeratin-5 and surfactant protein C (SPC). The method is A step of seeding MSCs on a substrate in which no other cell types are present; and A process of differentiating MSCs into lung cells expressing at least one epithelial marker by exposing a substrate seeded with MSCs to a growth medium containing retinoic acid and human epidermal growth factor. Includes, The MSC is a bone marrow-derived MSC (BM-MSC). The aforementioned population of lung epithelial cells.

2. A population of lung epithelial cells according to claim 1, wherein the lung epithelial cells are type II alveolar epithelial cells.

3. A population of lung epithelial cells according to claim 1, wherein lung epithelial cells are seeded on a substrate.

4. A population of lung epithelial cells according to claim 3, wherein the substrate is decellularized lung tissue.

5. A population of lung epithelial cells according to claim 3, wherein the substrate is a coating containing an extracellular matrix.

6. A population of lung epithelial cells according to claim 5, wherein the extracellular matrix comprises one or more of human ECM, laminin, fibronectin, collagen IV, and collagen I.

7. A population of lung epithelial cells according to claim 1, including genetically modified cells.

8. The population of lung epithelial cells according to claim 7, wherein the genetically modified cells are lung epithelial cells that have been genetically modified to express a therapeutic gene.

9. A composition for reducing or treating lung abnormalities in mammals, The composition comprising a therapeutically effective amount of a population of lung epithelial cells according to any one of claims 1 to 8.

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