Activin-Directed Differentiation of Human Embryonic Stem Cells Differentially Modulates Alveolar Epithelial Wound Repair via Paracrine Mechanism

Khondoker M. Akram, Monica A. Spiteri, Nicholas R. Forsyth

Institute for Science and Technology in Medicine, School of Postgraduate Medicine, Keele University, Stoke-on-Trent, UK.

DOI: 10.4236/scd.2014.43008                

Abstract

Differentiated embryonic stem cells (ESC) can ameliorate lung inflammation and fibrosis in animal lung injury models; therefore, ESC, or their products, could be candidates for regenerative therapy for incurable lung diseases, such as idiopathic pulmonary fibrosis (IPF). In this study, we have investigated the paracrine effect of differentiated and undifferentiated human ESC on alveolar epithelial cell (AEC) wound repair. hESC line, SHEF-2 cells were differentiated with Activin treatment for 22 days in an embryoid body (EB) suspension culture. Conditioned media (CM) which contain cell secretory factors were collected at different time points of differentiation. CM were then tested onin vitro wound repair model with human type II AEC line, A549 cells (AEC). Our study demonstrated that CM originated from undifferentiated hESC significantly inhibited AEC wound repair when compared to the control. Whereas, CM originated from Activin-directed hESC differentiated cell population demonstrated a differential reparative effect on AEC wound repair model. CM obtained from Day-11 of differentiation significantly enhanced AEC wound repair in comparison to CM collected from pre- and post-Day-11 of differentiation. Day-11 CM enhanced AEC wound repair through significant stimulation of cell migration and cell proliferation. RT-PCR and immunocytochemistry confirmed that Day-11 CM was originated form a mixed population of endodermal/mesodermal differentiated hESC. This report suggests a putative paracrine-mediated epithelial injury healing mechanism by hESC secreted products, which is valuable in the development of novel stem cell-based therapeutic strategies.

Keywords

Embryonic Stem Cells, Alveolar Epithelial Wound Repair, Idiopathic Pulmonary Fibrosis (IPF), Embryonic Stem Cell-Mediated Paracrine Mechanism, Activin-Directed hESC Differentiation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Raghu, G., Collard, H.R., Egan, J.J., et al. (2011) ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Statement: Idiopathic Pulmonary Fibrosis: Evidence-Based Guidelines for Diagnosis and Management. American Journal of Respiratory and Critical Care Medicine, 183, 788-824. http://dx.doi.org/10.1164/rccm.2009-040GL
[2]	Selman, M., King, T.E. and Pardo, A. (2001) American Thoracic Society, European Respiratory Society, American College of Chest Physicians. Idiopathic Pulmonary Fibrosis: Prevailing and Evolving Hypotheses about Its Pathogenesis and Implications for Therapy. Annals of Internal Medicine, 134, 136-151. http://dx.doi.org/10.7326/0003-4819-134-2-200101160-00015.
[3]	Strieter, R.M. (2005) Pathogenesis and Natural History of Usual Interstitial Pneumonia: The Whole Story or the Last Chapter of a Long Novel. Chest, 128, 526S-532S. http://dx.doi.org/10.1378/chest.128.5_suppl_1.526S
[4]	Mason, R.J. (2006) Biology of Alveolar Type II Cells. Respirology, Suppl. 11, S12-S15. http://dx.doi.org/10.1111/j.1440-1843.2006.00800.x
[5]	Zahm, J.M., Chevillard, M. and Puchelle, E. (1991) Wound Repair of Human Surface Respiratory Epithelium. American Journal of Respiratory Cell and Molecular Biology, 5, 242-248. http://dx.doi.org/10.1165/ajrcmb/5.3.242
[6]	Puchelle, E., Zahm, J.M., Tournier, J.M., et al. (2006) Airway Epithelial Repair, Regeneration, and Remodeling after Injury in Chronic Obstructive Pulmonary Disease. Proceedings of the American Thoracic Society, 3, 726-733. http://dx.doi.org/10.1513/pats.200605-126SF
[7]	Crosby, L.M. and Waters, C.M. (2010) Epithelial Repair Mechanisms in the Lung. AJP—Lung Cellular and Molecular Physiology, 298, L715-L731.
[8]	Corrin, B., Dewar, A., Rodriguez-Roisin, R., et al. (1985) Fine Structural Changes in Cryptogenic Fibrosing Alveolitis and Asbestosis. The Journal of Pathology, 147, 107-119. http://dx.doi.org/10.1002/path.1711470206
[9]	Uhal, B.D., Joshi, I., Hughes, W.F., et al. (1998) Alveolar Epithelial Cell Death Adjacent to Underlying Myofibroblasts in Advanced Fibrotic Human Lung. American Journal of Physiology—Lung Cellular and Molecular Physiology, 275, L1192-L1199.
[10]	Kuwano, K., Miyazaki, H., Hagimoto, N., et al. (1999) The Involvement of Fas-Fas Ligand Pathway in Fibrosing Lung Diseases. American Journal of Respiratory Cell and Molecular Biology, 20, 53-60. http://dx.doi.org/10.1165/ajrcmb.20.1.2941
[11]	Barbas-Filho, J.V., Ferreira, M.A., Sesso, A., et al. (2001) Evidence of Type II Pneumocyte Apoptosis in the Pathogenesis of Idiopathic Pulmonary Fibrosis (IFP)/Usual Interstitial Pneumonia (UIP). Journal of Clinical Pathology, 54, 132-138. http://dx.doi.org/10.1136/jcp.54.2.132
[12]	Akram, K.M., Samad, S., Spiteri, M., et al. (2013) Mesenchymal Stem Cell Therapy and Lung Diseases. Advances in Biochemical Engineering/Biotechnology, 130, 105-29. http://dx.doi.org/10.1007/10_2012_140
[13]	Akram, K.M., Lomas, N.J., Spiteri, M.A., et al. (2013) Club Cells Inhibit Alveolar Epithelial Wound Repair via TRAILDependent Apoptosis. European Respiratory Journal, 41, 683-694. http://dx.doi.org/10.1183/09031936.00213411
[14]	Selman, M. and Pardo, A. (2002) Idiopathic Pulmonary Fibrosis: An Epithelial/Fibroblastic Cross-Talk Disorder. Respiratory Research, 3, 3. http://dx.doi.org/10.1186/rr175
[15]	Odajima, N., Betsuyaku, T., Nasuhara, Y., et al. (2007) Loss of Caveolin-1 in Bronchiolization in Lung Fibrosis. Journal of Histochemistry & Cytochemistry, 55, 899-909. http://dx.doi.org/10.1369/jhc.7A7203.2007
[16]	Chilosi, M., Poletti, V., Murer, B., Lestani, M., Cancellieri, A., Montagna, L., et al. (2002) Abnormal Re-Epithelialization and Lung Remodeling in Idiopathic Pulmonary Fibrosis: The Role of ?N-p63. Laboratory Investigation, 82, 1335-1345. http://dx.doi.org/10.1097/01.LAB.0000032380.82232.67
[17]	Cisneros, J., Hagood, J., Checa, M., Ortiz-Quintero, B., Negreros, M., Herrera, I., et al. (2012) Hypermethylation-Mediated Silencing of p14ARF in Fibroblasts from Idiopathic Pulmonary Fibrosis. AJP, Lung Cellular and Molecular Physiology, 303, L295-L303.
[18]	Hogaboam, C.M., Murray, L. and Martinez, F.J. (2012) Epigenetic Mechanisms through Which Toll-Like Receptor-9 Drives Idiopathic Pulmonary Fibrosis Progression. Proceedings of the American Thoracic Society, 9, 172-176. http://dx.doi.org/10.1513/pats.201201-002AW
[19]	Rabinovich, E.I., Kapetanaki, M.G., Steinfeld, I., Gibson, K.F., Pandit, K.V., Yu, G., et al. (2012) Global Methylation Patterns in Idiopathic Pulmonary Fibrosis. PLoS ONE, 7, Article ID: e33770. http://dx.doi.org/10.1371/journal.pone.0033770
[20]	Weiss, D.J. and Finck, C. (2010) Embryonic Stem Cells and Repair of Lung Injury. Molecular Therapy, 18, 460-461. http://dx.doi.org/10.1038/mt.2010.8
[21]	Evans, M.J. and Kaufman, M.H. (1981) Establishment in Culture of Pluripotential Cells from Mouse Embryos. Nature, 292, 154-156. http://dx.doi.org/10.1038/292154a0
[22]	Kaufman, M.H., Robertson, E.J., Handyside, A.H. and Evans, M.J. (1983) Establishment of Pluripotential Cell Lines from Haploid Mouse Embryos. Journal of Embryology & Experimental Morphology, 73, 249-261.
[23]	Martin, G.R. (1981) Isolation of a Pluripotent Cell Line from Early Mouse Embryos Cultured in Medium Conditioned by Teratocarcinoma Stem Cells. Proceedings of the National Academy of Sciences of the United States of America, 78, 7634-7638. http://dx.doi.org/10.1073/pnas.78.12.7634
[24]	Mummery, C., Ward, D., van den Brink, C.E., Bird, S.D., Doevendans, P.A., Opthof, T., et al. (2002) Cardiomyocyte Differentiation of Mouse and Human Embryonic Stem Cells. Journal of Anatomy, 200, 233-242. http://dx.doi.org/10.1046/j.1469-7580.2002.00031.x
[25]	Mummery, C., Oostwaard, D.W., Doevendans, P., Spijker, R., van den Brink, S., Hassink, R., et al. (2003) Differentiation of Human Embryonic Stem Cells to Cardiomyocytes: Role of Coculture with Visceral Endoderm-Like Cells. Circulation, 107, 2733-2740. http://dx.doi.org/10.1161/01.CIR.0000068356.38592.68
[26]	Ying, Q., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003) Conversion of Embryonic Stem Cells into Neuroectodermal Precursors in Adherent Monoculture. Nature Biotechnology, 21, 183-186. http://dx.doi.org/10.1038/nbt780
[27]	Shin, S., Dalton, S. and Stice, S.L. (2005) Human Motor Neuron Differentiation from Human Embryonic Stem Cells. Stem Cells and Development, 14, 266-269. http://dx.doi.org/10.1089/scd.2005.14.266
[28]	Abranches, E., Silva, M., Pradier, L., Schulz, H., Hummel, O., Henrique, D., et al. (2009) Neural Differentiation of Embryonic Stem Cells in Vitro: A Road Map to Neurogenesis in the Embryo. PLoS ONE, 4, Article ID: e6286. http://dx.doi.org/10.1371/journal.pone.0006286
[29]	D’Amour, K.A., Agulnick, A.D., Eliazer, S., Kelly, O.G., Kroon, E. and Baetge, E.E. (2005) Efficient Differentiation of Human Embryonic Stem Cells to Definitive Endoderm. Nature Biotechnology, 23, 1534-1541. http://dx.doi.org/10.1038/nbt1163
[30]	Kubo, A., Shinozaki, K., Shannon, J.M., Kouskoff, V., Kennedy, M., Woo, S., et al. (2004) Development of Definitive Endoderm from Embryonic Stem Cells in Culture. Development, 131, 1651-1662. http://dx.doi.org/10.1242/dev.01044
[31]	McLean, A.B., D’Amour, K.A., Jones, K.L., Krishnamoorthy, M., Kulik, M.J., Reynolds, D.M., et al. (2007) Activin A Efficiently Specifies Definitive Endoderm from Human Embryonic Stem Cells Only When Phosphatidylinositol 3-Kinase Signaling Is Suppressed. Stem Cells, 25, 29-38. http://dx.doi.org/10.1634/stemcells.2006-0219
[32]	Colman, A. (2004) Making New Beta Cells from Stem Cells. Seminars in Cell & Developmental Biology, 15, 337-345. http://dx.doi.org/10.1016/j.semcdb.2004.02.003
[33]	Stoffel, M., Vallier, L. and Pedersen, R.A. (2004) Navigating the Pathway from Embryonic Stem Cells to Beta Cells. Seminars in Cell & Developmental Biology, 15, 327-336. http://dx.doi.org/10.1016/j.semcdb.2004.02.002
[34]	Hamazaki, T., Iiboshi, Y., Oka, M., Papst, P.J., Meacham, A.M., Zon, L.I., et al. (2001) Hepatic Maturation in Differentiating Embryonic Stem Cells in Vitro. FEBS Letters, 497, 15-19. http://dx.doi.org/10.1016/S0014-5793(01)02423-1
[35]	Jones, E.A., Tosh, D., Wilson, D.I., Lindsaya, S. and Forrester, L.M. (2002) Hepatic Differentiation of Murine Embryonic Stem Cells. Experimental Cell Research, 272, 15-22. http://dx.doi.org/10.1006/excr.2001.5396
[36]	Yamada, T., Yoshikawa, M., Kanda, S., Kato, Y., Nakajima, Y., Ishizaka, S., et al. (2002) In Vitro Differentiation of Embryonic Stem Cells into Hepatocyte-Like Cells Identified by Cellular Uptake of Indocyanine Green. Stem Cells, 20, 146-154. http://dx.doi.org/10.1634/stemcells.20-2-146
[37]	Yamada, T., Yoshikawa, M., Takaki, M., Torihashi, S., Kato, Y., Nakajima, Y., et al. (2002) In Vitro Functional GutLike Organ Formation from Mouse Embryonic Stem Cells. Stem Cells, 20, 41-49. http://dx.doi.org/10.1634/stemcells.20-1-41
[38]	Ali, N.N., Edgar, A.J., Samadikuchaksaraei, A., Timson, C.M., Romanska, H.M., Polak, J.M., et al. (2002) Derivation of Type II Alveolar Epithelial Cells from Murine Embryonic Stem Cells. Tissue Engineering, 8, 541-550. http://dx.doi.org/10.1089/107632702760240463
[39]	Rippon, H.J., Polak, J.M., Qin, M. and Bishop, A.E. (2006) Derivation of Distal Lung Epithelial Progenitors from Murine Embryonic Stem Cells Using a Novel Three-Step Differentiation Protocol. Stem Cells, 24, 1389-1398. http://dx.doi.org/10.1634/stemcells.2005-0465
[40]	Wang, D., Morales, J.E., Calame, D.G., Alcorn, J.L. and Wetsel, R.A. (2010) Transplantation of Human Embryonic Stem Cell-Derived Alveolar Epithelial Type II Cells Abrogates Acute Lung Injury in Mice. Molecular Therapy, 18, 625-634. http://dx.doi.org/10.1038/mt.2009.317
[41]	Roszell, B., Mondrinos, M.J., Seaton, A., Simons, D.M., Koutzaki, S.H., Fong, G.H., et al. (2009) Efficient Derivation of Alveolar Type II Cells from Embryonic Stem Cells for in Vivo Application. Tissue Engineering Part A, 15, 3351-3365. http://dx.doi.org/10.1089/ten.tea.2008.0664
[42]	Geiser, T., Jarreau, P.H., Atabai, K. and Matthay, M.A. (2000) Interleukin-1beta Augments in Vitro Alveolar Epithelial Repair. AJP, Lung Cellular and Molecular Physiology, 279, L1184-L1190.
[43]	Atabai, K., Ishigaki, M., Geiser, T., Ueki, I., Matthay, M.A. and Ware, L.B. (2002) Keratinocyte Growth Factor Can Enhance Alveolar Epithelial Repair by Nonmitogenic Mechanisms. AJP, Lung Cellular and Molecular Physiology, 283, L163-L169.
[44]	Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., et al. (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts. Science, 282, 1145-1147. http://dx.doi.org/10.1126/science.282.5391.1145
[45]	Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. and Bongso, A. (2000) Embryonic Stem Cell Lines from Human Blastocysts: Somatic Differentiation in Vitro. Nature Biotechnology, 18, 399-404. http://dx.doi.org/10.1038/74447
[46]	Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., et al. (2003) Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells. Cell, 113, 643-655. http://dx.doi.org/10.1016/S0092-8674(03)00392-1
[47]	Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., et al. (2003) The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells. Cell, 113, 631-642. http://dx.doi.org/10.1016/S0092-8674(03)00393-3
[48]	Abe, K., Niwa, H., Iwase, K., Takiguchi, M., Mori, M., Abé, S.I., et al. (1996) Endoderm-Specific Gene Expression in Embryonic Stem Cells Differentiated to Embryoid Bodies. Experimental Cell Research, 229, 27-34. http://dx.doi.org/10.1006/excr.1996.0340
[49]	Bruce, S.J., Gardiner, B.B., Burke, L.J., Gongora, M.M., Grimmond, S.M. and Perkins, A.C. (2007) Dynamic Transcription Programs during ES Cell Differentiation towards Mesoderm in Serum versus Serum-FreeBMP4 Culture. BMC Genomics, 8, 365. http://dx.doi.org/10.1186/1471-2164-8-365
[50]	Pevny, L.H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998) A Role for SOX1 in Neural Determination. Development, 125, 1967-1978.
[51]	Ortiz, L.A., Dutreil, M., Fattman, C., Pandey, A.C., Torres, G., Go, K., et al. (2007) Interleukin 1 Receptor Antagonist Mediates the Antiinflammatory and Antifibrotic Effect of Mesenchymal Stem Cells during Lung Injury. Proceedings of the National Academy of Sciences of the United States of America, 104, 11002-11007. http://dx.doi.org/10.1073/pnas.0704421104
[52]	Zhen, G., Liu, H., Gu, N., Zhang, H., Xu, Y. and Zhang, Z. (2008) Mesenchymal Stem Cells Transplantation Protects against Rat Pulmonary Emphysema. Frontiers in Bioscience, 13, 3415-3422. http://dx.doi.org/10.2741/2936
[53]	Burdon, T.J., Paul, A., Noiseux, N., Prakash, S. and Shum-Tim, D. (2011) Bone Marrow Stem Cell Derived Paracrine Factors for Regenerative Medicine: Current Perspectives and Therapeutic Potential. Bone Marrow Research, 2011, Article ID: 207326. http://dx.doi.org/10.1155/2011/207326
[54]	Baber, S.R., Deng, W., Master, R.G., Bunnell, B.A., Taylor, B.K., Murthy, S.N., et al. (2007) Intratracheal Mesenchymal Stem Cell Administration Attenuates Monocrotaline-Induced Pulmonary Hypertension and Endothelial Dysfunction. AJP, Heart and Circulatory Physiology, 292, H1120-H1128.
[55]	Nemeth, K., Keane-Myers, A., Brown, J.M., Metcalfec, D.D., Gorhamd, J.D., Bundoc, V.G., et al. (2010) Bone Marrow Stromal Cells Use TGF-Beta to Suppress Allergic Responses in a Mouse Model of Ragweed-Induced Asthma. Proceedings of the National Academy of Sciences of the United States of America, 107, 5652-5657. http://dx.doi.org/10.1073/pnas.0910720107
[56]	Banerjee, E.R., Laflamme, M.A., Papayannopoulou, T., Kahn, M., Murry, C.E. and Henderson Jr., W.R. (2012) Human Embryonic Stem Cells Differentiated to Lung Lineage-Specific Cells Ameliorate Pulmonary Fibrosis in a Xenograft Transplant Mouse Model. PLoS ONE, 7, Article ID: e33165. http://dx.doi.org/10.1371/journal.pone.0033165
[57]	Crisostomo, P.R., Abarbanell, A.M., Wang, M., Lahm, T., Wang, Y. and Meldrum, D.R. (2008) Embryonic Stem Cells Attenuate Myocardial Dysfunction and Inflammation after Surgical Global Ischemia via Paracrine Actions. AJP, Heart and Circulatory Physiology, 295, H1726-H1735.
[58]	LaFramboise, W.A., Petrosko, P., Krill-Burger, J.M., Morris, D.R., McCoya, A.R., Scalise, D., et al. (2010) Proteins Secreted by Embryonic Stem Cells Activate Cardiomyocytes through Ligand Binding Pathways. Journal of Proteomics, 73, 992-1003. http://dx.doi.org/10.1016/j.jprot.2009.12.013
[59]	Singla, D.K. and McDonald, D.E. (2007) Factors Released from Embryonic Stem Cells Inhibit Apoptosis of H9c2 Cells. AJP, Heart and Circulatory Physiology, 293, H1590-H1595.
[60]	Singla, D.K., Singla, R.D. and McDonald, D.E. (2008) Factors Released from Embryonic Stem Cells Inhibit Apoptosis in H9c2 Cells through PI3K/Akt but Not ERK Pathway. AJP, Heart and Circulatory Physiology, 295, H907-H913.
[61]	Lu, X., Chen, D., Liu, Z., Li, C., Liu, Y., Zhou, J., et al. (2010) Enhanced Survival in Vitro of Human Corneal Endothelial Cells Using Mouse Embryonic Stem Cell Conditioned Medium. Molecular Vision, 16, 611-622.
[62]	Sisson, T.H., Mendez, M., Choi, K., Subbotina, N., Courey, A., Cunningham, A., et al. (2010) Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis. American Journal of Respiratory and Critical Care Medicine, 181, 254-263. http://dx.doi.org/10.1164/rccm.200810-1615OC
[63]	Buckley, S., Shi, W., Carraro, G., Sedrakyan, S., Sacco, S.D., Driscoll, B.A., et al. (2011) The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors. American Journal of Respiratory Cell and Molecular Biology, 45, 1212-1221. http://dx.doi.org/10.1165/rcmb.2010-0325OC
[64]	Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D., et al. (2001) Feeder-Free Growth of Undifferentiated Human Embryonic Stem Cells. Nature Biotechnology, 19, 971-974. http://dx.doi.org/10.1038/nbt1001-971
[65]	Plumb, J.A., Milroy, R. and Kaye, S.B. (1989) Effects of the pH Dependence of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-Tetrazolium Bromide-Formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-Based Assay. Cancer Research, 49, 4435-4440.