Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

An overview and synopsis of techniques for directing stem cell differentiation in vitro

  • 343 Accesses

  • 52 Citations


The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.

This is a preview of subscription content, log in to check access.


  1. Ahrens M, Ankenbauer T, Schröder D, Hollnagel A, Mayer H, Gross G (1993) Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol 12:871–880

  2. Akita J, Takahashi M, Hojo M, Nishida A, Haruta M, Honda Y (2002) Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment. Brain Res 954:286–293

  3. Alberts B (2002) Molecular biology of the cell. Garland Science, New York

  4. Allen RG, Balin AK (1989) Oxidative influence on development and differentiation: an overview of a free radical theory of development. Free Radic Biol Med 6:6 631–661

  5. Almeida-Porada G, Brown RL, MacKintosh FR, Zanjani ED (2003) Evaluation of serum-free culture conditions able to support the ex vivo expansion and engraftment of human hematopoietic stem cells in the human-to-sheep xenograft model. J Hematother Stem Cell Res 9:683–693

  6. Altman GH, Horan RL, Martin I, Farhadi J, Stark PR, Volloch V, Richmond JC, Vunjak-Novakovic G, Kaplan DL (2002) Cell differentiation by mechanical stress. FASEB J 16:270–272

  7. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68:2150–2156

  8. An YH, Wan H, Zhang ZS, Wang HY, Gao ZX, Sun MZ, Wang ZC (2003) Effect of rat Schwann cell secretion on proliferation and differentiation of human neural stem cells. Biomed Environ Sci 16:90–94

  9. Andrews PW (1998) Teratocarcinomas and human embryology: pluripotent human EC cell lines. Review article. APMIS 106:158–167; discussion 167–168

  10. Angele P, Kujat R, Nerlich M, Yoo J, Goldberg V, Johnstone B (1999) Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge. Tissue Eng 5:545–554

  11. Awad HA, Butler DL, Boivin GP, Smith FN, Malaviya P, Huibregtse B, Caplan AI (1999) Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 5:267–277

  12. Badorff C, Brandes RP, Popp R, Rupp S, Urbich C, Aicher A, Fleming I, Busse R, Zeiher AM, Dimmeler S (2003) Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation 107:1024–1032

  13. Bernhard H, Lohmann M, Batten WY, Metzger J, Löhr HF, Peschel C, zum Büschenfelde KM, Rose-John S (2000) The gp130-stimulating designer cytokine hyper-IL-6 promotes the expansion of human hematopoietic progenitor cells capable to differentiate into functional dendritic cells. Exp Hematol 28:365–372

  14. Brazelton TR, Rossi FM, Keshet GI, Blau HM (2000) From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290:1775–1779

  15. Buttery LD, Bourne S, Xynos JD, Wood H, Hughes FJ, Hughes SP, Episkopou V, Polak JM (2001) Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng 7:89–99

  16. Carystinos GD, Alaoui-Jamali MA, Phipps J, Yen L, Batist G (2001) Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells. Cancer Chemother Pharmacol 47:126–132

  17. Chen SS, Revoltella RP, Papini S, Michelini M, Fitzgerald W, Zimmerberg J, Margolis L (2003) Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems. Stem Cells 21:281–295

  18. Cheng L, Hammond H, Ye Z, Zhan X, Dravid G (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21:131–142

  19. Chipperfield H, Bedi KS, Cool SM, Nurcombe V (2002) Heparan sulfates isolated from adult neural progenitor cells can direct phenotypic maturation. Int J Dev Biol 46:661–670

  20. Chute JP, Saini AA, Chute DJ, Wells MR, Clark WB, Harlan DM, Park J, Stull MK, Civin C, Davis TA (2002) Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow. Blood 100:4433–4439

  21. Comper WP (1996) Extracellular matrix, vol 2, molecular components and interactions. Harwood Academic, Amsterdam

  22. Condorelli G, Borello U, De Angelis L, Latronico M, Sirabella D, Coletta M, Galli R, Balconi G, Follenzi A, Frati G, Cusella De Angelis MG, Gioglio L, Amuchastegui S, Adorini L, Naldini L, Vescovi A, Dejana E, Cossu G (2001) Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration. Proc Natl Acad Sci U S A 98:10733–10738

  23. Conget PA, Minguell JJ (2000) Adenoviral-mediated gene transfer into ex vivo expanded human bone marrow mesenchymal progenitor cells. Exp Hematol 28:382–390

  24. Czyz J, Wobus A (2001) Embryonic stem cell differentiation: the role of extracellular factors. Differentiation 68:167–174

  25. Davidson RL, Gerald PS (1977) Induction of mammalian somatic cell hybridization by polyethylene glycol. Methods Cell Biol 15:325–338

  26. Dowling-Warriner CV, Trosko JE (2000) Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway. Neuroscience 95:859–868

  27. Dragoo JL, Choi JY, Lieberman JR, Huang J, Zuk PA, Zhang J, Hedrick MH, Benhaim P (2003) Bone induction by BMP-2 transduced stem cells derived from human fat. J Orthop Res 21:622–629

  28. Eremenko T, Esposito C, Pasquarelli A, Pasquali E, Volpe P (1997) Cell-cycle kinetics of Friend erythroleukemia cells in a magnetically shielded room and in a low-frequency/low-intensity magnetic field. Bioelectromagnetics 18:58–66

  29. Fedyk ER, Phipps RP (1994) Reactive oxygen species and not lipoxygenase products are required for mouse B-lymphocyte activation and differentiation. Int J Immunopharmacol 16:7 533–546

  30. Ferrari G, Cusella De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F (1998) Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279:1528–1530

  31. Flores-Guzmán P, Gutiérrez-Rodríguez M, Mayani H (2002) In vitro proliferation, expansion, and differentiation of a CD34+ cell-enriched hematopoietic cell population from human umbilical cord blood in response to recombinant cytokines. Arch Med Res 33:107–114

  32. Fukuda K (2002) Molecular characterization of regenerated cardiomyocytes derived from adult mesenchymal stem cells. Congenit Anom Kyoto 42:1–9

  33. Fulco M, Schiltz RL, Iezzi S, King MT, Zhao P, Kashiwaya Y, Hoffman E, Veech RL, Sartorelli V (2003) Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Mol Cell 12:51–62

  34. Galli R, Borello U, Gritti A, Minasi MG, Bjornson C, Coletta M, Mora M, De Angelis MG, Fiocco R, Cossu G, Vescovi AL (2000) Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci 3:986–991

  35. Gibbs SM (2002) Regulation of neuronal proliferation and differentiation by nitric oxide. Mol Neurobiol 27:107–120

  36. Giovino MA, Down JD, Jackson JD, Sykes M, Monroy RL, White-Scharf ME (2002) Porcine hematopoiesis on primate stroma in long-term cultures: enhanced growth with neutralizing tumor necrosis factor-alpha and tumor growth factor-beta antibodies. Transplantation 73:723–731

  37. Gitelman SE, Kirk M, Ye JQ, Filvaroff EH, Kahn AJ, Derynck R (1995) Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells. Cell Growth Differ 6:827–836

  38. Goldman RD, Pollack R, Hopkins NH (1973) Preservation of normal behavior by enucleated cells in culture. Proc Natl Acad Sci U S A 70:750–754

  39. Goldsborough MD, Tilkins ML, Price PJ, Lobo-Alfonso J, Morrison JR, Stevens ME (1998) Serum-free culture of murine embryonic stem (ES) cells. Focus 20:8–12

  40. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW (2002) Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 607–612

  41. Gratsch TE, O’Shea KS (2002) Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells. Dev Biol 245:83–94

  42. Griffith M, Hakim M, Shimmura S, Watsky MA, Li F, Carlsson D, Doillon CJ, Nakamura M, Suuronen E, Shinozaki N, Nakata K, Sheardown H (2002) Artificial human corneas: scaffolds for transplantation and host regeneration. Cornea 21:S54–S61

  43. Gronthos S, Simmons PJ, Graves SE, Robey PG (2001) Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone 28:174–181

  44. Guan K, Chang H, Rolletschek A, Wobus AM (2001) Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res 305:171–176

  45. Gulbins H, Meiser BM, Reichenspurner H, Reichart B (2002) Cell transplantation—a potential therapy for cardiac repair in the future? Heart Surg Forum 5:E28–34

  46. Gurevich O, Vexler A, Marx G, Prigozhina T, Levdansky L, Slavin S, Shimeliovich I, Gorodetsky R (2002) Fibrin microbeads for isolating and growing bone marrow-derived progenitor cells capable of forming bone tissue. Tissue Eng 8:661–672

  47. Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401:390–394

  48. Håkelien AM, Collas P (2002) Novel approaches to transdifferentiation. Cloning Stem Cells 4:379–387

  49. Håkelien AM, Landsverk HB, Robl JM, Skålhegg BS, Collas P (2002) Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat Biotechnol 20:460–466

  50. Hall FL, Han B, Kundu RK, Yee A, Nimni ME, Gordon EM (2001) Phenotypic differentiation of TGF-beta1-responsive pluripotent premesenchymal prehematopoietic progenitor (P4 stem) cells from murine bone marrow. J Hematother Stem Cell Res 10:261–271

  51. Halliwell JV, Horne AL (1998) Evidence for enhancement of gap junctional coupling between rat island of Calleja granule cells in vitro by the activation of dopamine D3 receptors. J Physiol 506:175–194

  52. Hartig M, Joos U, Wiesmann HP (2000) Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro. Eur Biophys J 29:499–506

  53. Herzenberg LA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48:1819–1827

  54. Itskovitz Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6:88–95

  55. Iwakura Y, Nozaki M, Asano M, Yoshida MC, Tsukada Y, Hibi N, Ochiai A, Tahara E, Tosu M, Sekiguchi T (1985) Pleiotropic phenotypic expression in cybrids derived from mouse teratocarcinoma cells fused with rat myoblast cytoplasts. Cell 43:777–791

  56. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, Goodell MA (2001) Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107:1395–1402

  57. Jin D, Zeng WS, Pei GX, Qi FJ, Wei KH, Wang K, Chen B (2002) Expression of human bone morphogenetic protein 7 mRNA after the gene transfection in rabbit bone marrow-derived mesenchymal stem cells. Di Yi Jun Yi Da Xue Xue Bao 22:222–225

  58. Jurga M, Buzańska L (2003) The influence of Id1 protein on human stem cells differentiation into neuronal pathway. J Neurochem 85:Suppl 2:33

  59. Kallos MS, Sen A, Behie LA (2003) Large-scale expansion of mammalian neural stem cells: a review. Med Biol Eng Comput 41:271–282

  60. Kaneko Y, Ichikawa M, Kurimoto Y, Ohta K, Yoshimura N (2003) Neuronal differentiation of hippocampus-derived neural stem cells cultured in conditioned medium of embryonic rat retina. Ophthalmic Res 35:268–275

  61. Kannan MS, Daniel EE (1978) Formation of gap junctions by treatment in vitro with potassium conductance blockers. J Cell Biol 78:338–348

  62. Kapur S, Baylink DJ, William Lau KH (2003) Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone 32:241–251

  63. Karadag A, Scutt AM, Croucher PI (2000) Human myeloma cells promote the recruitment of osteoblast precursors: mediation by interleukin-6 and soluble interleukin-6 receptor. J Bone Miner Res 15:1935–1943

  64. Kato S, Sugiura N, Kimata K, Kujiraoka T, Toyoda J, Akamatsu N (1995) Chondroitin sulfate immobilized onto culture substrates modulates DNA synthesis, tyrosine aminotransferase induction, and intercellular communication in primary rat hepatocytes. Cell Struct Funct 20:199–209

  65. Kim BJ, Seo JH, Bubien JK, Oh YS (2002) Differentiation of adult bone marrow stem cells into neuroprogenitor cells in vitro. Neuroreport 13:1185–1188

  66. Koffler L, Roshong S, Kyu Park I, Cesen-Cummings K, Thompson DC, Dwyer-Nield LD, Rice P, Mamay C, Malkinson AM, Ruch RJ (2000) Growth inhibition in G(1) and altered expression of cyclin D1 and p27(kip-1 ) after forced connexin expression in lung and liver carcinoma cells. J Cell Biochem 79:347–354

  67. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ (2001) Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105:369–377

  68. Kyrylenko S, Kyrylenko O, Suuronen T, Salminen A (2003) Differential regulation of the Sir2 histone deacetylase gene family by inhibitors of class I and II histone deacetylases. Cell Mol Life Sci 60:1990–1997

  69. Lako M, Lindsay S, Lincoln J, Cairns PM, Armstrong L, Hole N (2001) Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation. Mech Dev 103:49–59

  70. Lennon DP, Haynesworth SE, Young RG, Dennis JE, Caplan AI (1995) A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells. Exp Cell Res 219:211–222

  71. Li F, Lu S, Vida L, Thomson JA, Honig GR (2001a) Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. Blood 98:335–342

  72. Li Y, Ma T, Kniss DA, Yang ST, Lasky LC (2001b) Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro simulation of the marrow microenvironment. J Hematother Stem Cell Res 10:355–368

  73. Liu Z, Martin LJ (2003) Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human. J Comp Neurol 459:368–391

  74. Liu S, Qu Y, Stewart TJ, Howard MJ, Chakrabortty S, Holekamp TF, McDonald JW (2000) Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci U S A 97:6126–6131

  75. Liu CY, Apuzzo ML, Tirrell DA (2003) Engineering of the extracellular matrix: working toward neural stem cell programming and neurorestoration—concept and progress report. Neurosurgery 52:1154–1165; discussion 1165–1167

  76. Lohmann CH, Schwartz Z, Liu Y, Guerkov H, Dean DD, Simon B, Boyan BD (2000) Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production. J Orthop Res 18:637–646

  77. Lou S, Gu P, Chen F, He C, Wang M, Lu C (2003) The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats. Brain Res 968:114–121

  78. Lucas JJ, Kates JR (1976) The construction of viable nuclear-cytoplasmic hybrid cells by nuclear transplantation. Cell 7:397–405

  79. Mackenzie TC, Flake AW (2001) Multilineage differentiation of human MSC after in utero transplantation. Cytotherapy 3:403–405

  80. Madihally SV, Flake AW, Matthew HW (1999) Maintenance of CD34 expression during proliferation of CD34+ cord blood cells on glycosaminoglycan surfaces. Stem Cells 17:295–305

  81. Maruyama T, Umezawa A, Kusakari S, Kikuchi H, Nozaki M, Hata J (1996) Heat shock induces differentiation of human embryonal carcinoma cells into trophectoderm lineages. Exp Cell Res 224:123–127

  82. Matsuda N, Morita N, Matsuda K, Watanabe M (1998) Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res Commun 249:350–354

  83. McFarlane EH, Dawe GS, Marks M, Campbell IC (2000) Changes in neurite outgrowth but not in cell division induced by low EMF exposure: influence of field strength and culture conditions on responses in rat PC12 pheochromocytoma cells. Bioelectrochemistry 52:23–28

  84. Mie M, Endoh T, Yanagida Y, Kobatake E, Aizawa M (2003) Induction of neural differentiation by electrically stimulated gene expression of NeuroD2. J Biotechnol 100:231–238

  85. Mobest D, Mertelsmann R, Henschler R (1998) Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells. Biotechnol Bioeng 60:3 341–347

  86. Mody N, Parhami F, Sarafian TA, Demer LL (2001) Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radic Biol Med 15 31:4 509–519

  87. Moreno Lopez B, Noval JA, Gonzalez Bonet LG, Estrada C (2000) Morphological bases for a role of nitric oxide in adult neurogenesis. Brain Res 30 869:244–250

  88. Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, de la Riviere AB, Passier R, Tertoolen L (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107:2733–2740

  89. Nagy K, Pasti G, Bene L, Zs Nagy I (1993) Induction of granulocytic maturation in HL-60 human leukemia cells by free radicals: a hypothesis of cell differentiation involving hydroxyl radicals. Free Radic Res Commun 19:1–15

  90. Nakayama T, Momoki Soga T, Inoue N (2003) Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons. Neurosci Res 46:241–249

  91. Noble M, Smith J, Power J, Mayer Proschel M (2003) Redox state as a central modulator of precursor cell function. Ann N Y Acad Sci 991:251–271

  92. Odorico JS, Kaufman DS, Thomson JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19:193–204

  93. Okazaki T, Kato Y, Tashima M, Sawada H, Uchino H (1988) Evidence of intracellular and trans-acting differentiation-inducing activity in human promyelocytic leukemia HL-60 cells: its possible involvement in process of cell differentiation from a commitment step to a phenotype-expression step. J Cell Physiol 134:261–268

  94. Oravecz K, Kalka D, Jeney F, Cantz M, Zs Nagy I (2002) Hydroxyl free radicals induce cell differentiation in SK-N-MC neuroblastoma cells. Tissue Cell 34:33–38

  95. Orive G, Gascon AR, Hernandez RM, Igartua M, Luis Pedraz J (2003) Cell microencapsulation technology for biomedical purposes: novel insights and challenges. Trends Pharmacol Sci 24:207–210

  96. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal Ginard B, Bodine DM, Leri A, Anversa P (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410:701–705

  97. O’Shea KS (2001) Directed differentiation of embryonic stem cells: genetic and epigenetic methods. Wound Repair Regen 9:443–459

  98. Palacios R, Golunski E, Samaridis J (1995) In vitro generation of hematopoietic stem cells from an embryonic stem cell line. Proc Natl Acad Sci U S A 92:7530–7534

  99. Paulson AF, Lampe PD, Meyer RA, TenBroek E, Atkinson MM, Walseth TF, Johnson RG (2000) Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking. J Cell Sci 113:3037–3049

  100. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP (1999) Bone marrow as a potential source of hepatic oval cells. Science 284:1168–1170

  101. Phillips BW, Vernochet C, Dani C (2003) Differentiation of embryonic stem cells for pharmacological studies on adipose cells. Pharmacol Res 47:263–268

  102. Pimentel RC, Yamada KA, Kléber AG, Saffitz JE (2002) Autocrine regulation of myocyte Cx43 expression by VEGF. Circ Res 90:671–677

  103. Pirkkala L, Alastalo TP, Nykanen P, Seppa L, Sistonen L (1999) Differentiation lineage-specific expression of human heat shock transcription factor 2. FASEB J 13:1089–1098

  104. Poulsom R, Alison MR, Forbes SJ, Wright NA (2002) Adult stem cell plasticity. J Pathol 197:441–456

  105. Poznansky MC, Evans RH, Foxall RB, Olszak IT, Piascik AH, Hartman KE, Brander C, Meyer TH, Pykett MJ, Chabner KT, Kalams SA, Rosenzweig M, Scadden DT (2000) Efficient generation of human T cells from a tissue-engineered thymic organoid. Nat Biotechnol 18:729–734

  106. Rangappa S, Entwistle JW, Wechsler AS, Kresh JY (2003a) Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg 26:124–132

  107. Rangappa S, Fen C, Lee EH, Bongso A, Wei ES (2003b) Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg 75:775–779

  108. Rathjen J, Haines BP, Hudson KM, Nesci A, Dunn S, Rathjen PD (2002) Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population. Development 129:2649–2661

  109. Reddy S, Rayburn H, von Melchner H, Ruley HE (1992) Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes. Proc Natl Acad Sci U S A 89:6721–6725

  110. Ren P, de Feijter AW, Paul DL, Ruch RJ (1994) Enhancement of liver cell gap junction protein expression by glucocorticoids. Carcinogenesis 15:1807–1813

  111. Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20:933–936

  112. Roche P, Goldberg HA, Delmas PD, Malaval L (1999) Selective attachment of osteoprogenitors to laminin. Bone 24:329–336

  113. Rogers JJ, Young HE, Adkison LR, Lucas PA, Black AC (1995) Differentiation factors induce expression of muscle, fat, cartilage, and bone in a clone of mouse pluripotent mesenchymal stem cells. Am Surg 61:231–236

  114. Romanello M, Moro L, Pirulli D, Crovella S, D’Andrea P (2001) Effects of cAMP on intercellular coupling and osteoblast differentiation. Biochem Biophys Res Commun 282:1138–1144

  115. Sasaki T, Takagi M, Soma T, Yoshida T (2002) 3D culture of murine hematopoietic cells with spatial development of stromal cells in nonwoven fabrics. Cytotherapy 4:285–291

  116. Sauer H, Rahimi G, Hescheler J, Wartenberg M (1999) Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells. J Cell Biochem 75:710–723

  117. Sauer H, Rahimi G, Hescheler J, Wartenberg M (2000) Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells. FEBS Lett 7 476:218–223

  118. Sauer H, Wartenberg M, Hescheler J (2001) Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11:173–186

  119. Schinstine M, Iacovitti L (1997) 5-Azacytidine and BDNF enhance the maturation of neurons derived from EGF-generated neural stem cells. Exp Neurol 144:315–325

  120. Schumm MA, Castellanos DA, Frydel BR, Sagen J (2002) Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture. Brain Res Dev Brain Res 137:115–125

  121. Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 95:13726–13731

  122. Siegel DL (2002) Selecting antibodies to cell-surface antigens using magnetic sorting techniques. Methods Mol Biol 178:219–226

  123. Sistonen L, Sarge KD, Phillips B, Abravaya K, Morimoto RI (1992) Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells. Mol Cell Biol 12:4104–4111

  124. Sligh JE, Levy SE, Waymire KG, Allard P, Dillehay DL, Nusinowitz S, Heckenlively JR, MacGregor GR, Wallace DC (2000) Maternal germ-line transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice. Proc Natl Acad Sci U S A 97:14461–14466

  125. Sohal RS, Allen RG, Nations C (1986) Oxygen free radicals play a role in cellular differentiation: an hypothesis. J Free Radic Biol Med 2:175–181

  126. Song HJ, Stevens CF, Gage FH (2002) Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 5:438–445

  127. Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A, Peister A, Wang MY, Prockop DJ (2003) Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci U S A 100:2397–2402

  128. Stewart R, Przyborski S (2002) Non-neural adult stem cells: tools for brain repair? Bioessays 24:708–713

  129. Stocco DM (1983) Rapid, quantitative isolation of mitochondria from rat liver using Ficoll gradients in vertical rotors. Anal Biochem 131:453–457

  130. Su HL, Malbon CC, Wang HY (1993) Increased expression of Gi alpha 2 in mouse embryo stem cells promotes terminal differentiation to adipocytes. Am J Physiol 265:C1729–1735

  131. Suzuki A, Iwama A, Miyashita H, Nakauchi H, Taniguchi H (2003) Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells. Development 130:13–24

  132. Takano N, Kawakami T, Kawa Y, Asano M, Watabe H, Ito M, Soma Y, Kubota Y, Mizoguchi M (2002) Fibronectin combined with stem cell factor plays an important role in melanocyte proliferation, differentiation and migration in cultured mouse neural crest cells. Pigment Cell Res 15:192–200

  133. Tarone G, Hirsch E, Brancaccio M, De Acetis M, Barberis L, Balzac F, Retta SF, Botta C, Altruda F, Silengo L, Retta F (2000) Integrin function and regulation in development. Int J Dev Biol 44:725–731

  134. Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416:542–545

  135. Tezuka K, Yasuda M, Watanabe N, Morimura N, Kuroda K, Miyatani S, Hozumi N (2002) Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res 17:231–239

  136. Thomson JA, Itskovitz Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

  137. Toquet J, Rohanizadeh R, Guicheux J, Couillaud S, Passuti N, Daculsi G, Heymann D (1999) Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic. J Biomed Mater Res 44:98–108

  138. Tosu M, Terasaki T, Iwakura Y, Yoshida M, Sekiguchi T (1988) Clonal isolation and characterization of myoblast-like reconstituted cells formed by fusion of karyoplasts from mouse teratocarcinoma cells with rat myoblast cytoplasts. Cell Struct Funct 13:249–266

  139. Trounson A (2002) Human embryonic stem cells: mother of all cell and tissue types. Reprod Biomed 4 Suppl 1:58–63

  140. Tun T, Miyoshi H, Ema H, Nakauchi H, Ohshima N (2000) New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments. ASAIO J 46:522–526

  141. Tun T, Miyoshi H, Aung T, Takahashi S, Shimizu R, Kuroha T, Yamamoto M, Ohshima N (2002) Effect of growth factors on ex vivo bone marrow cell expansion using three-dimensional matrix support. Artif Organs 26:333–339

  142. Uzan G, Prandini MH, Rosa JP, Berthier R (1996) Hematopoietic differentiation of embryonic stem cells: an in vitro model to study gene regulation during megakaryocytopoiesis. Stem Cells 14 Suppl 1:194–199

  143. Verfaillie CM (2002) Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 12:502–508

  144. Voermans C, Gerritsen WR, von dem Borne AE, van der Schoot CE (1999) Increased migration of cord blood-derived CD34+ cells, as compared to bone marrow and mobilized peripheral blood CD34+ cells across uncoated or fibronectin-coated filters. Exp Hematol 27:1806–1814

  145. Volloch V, Kaplan D (2002) Matrix-mediated cellular rejuvenation. Matrix Biol 21:533–543

  146. Walsh D, Li Z, Wu Y, Nagata K (1997) Heat shock and the role of the HSPs during neural plate induction in early mammalian CNS and brain development. Cell Mol Life Sci 53:198–211

  147. Wan H, An Y, Zhang Z, Zhang Y, Wang Z (2003) Differentiation of rat embryonic neural stem cells promoted by co-cultured Schwann cells. Chin Med J (Engl) 116:428–431

  148. Wang Y, Mehta PP (1995) Facilitation of gap-junctional communication and gap-junction formation in mammalian cells by inhibition of glycosylation. Eur J Cell Biol 67:285–296

  149. Wang JS, Shum Tim D, Chedrawy E, Chiu RC (2001) The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg 122:699–705

  150. Watanabe T, Nomura S, Oishi M (1985) Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells. Exp Cell Res 159:224–234

  151. Watanabe T, Nomura S, Kaneko T, Yamagoe S, Kamiya T, Oishi M (1988) Cytoplasmic factors involved in erythroid differentiation in mouse erythroleukemia (MEL) cells. Cell Differ Dev 25 Suppl:105–109

  152. Weissman IL (2000) Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287:1442–1446

  153. Wigler MH, Weinstein IB (1975) A preparative method for obtaining enucleated mammalian cells. Biochem Biophys Res Commun 63:669–674

  154. Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji GJ, Fleischmann B, Katus HA, Hescheler J, Franz WM (1997) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol 29:1525–1539

  155. Wong M, Tuan RS (1993) Nuserum, a synthetic serum replacement, supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture. In Vitro Cell Dev Biol Anim 29A:917–922

  156. Xia Y, Buja LM, Scarpulla RC, McMillin JB (1997) Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation. Proc Natl Acad Sci U S A 94:11399–11404

  157. Yamada T, Hashiguchi A, Fukushima S, Kakita Y, Umezawa A, Maruyama T, Hata J (2000) Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells. In Vitro Cell Dev Biol Anim 36:139–146

  158. Yamaguchi M, Hirayama F, Murahashi H, Azuma H, Sato N, Miyazaki H, Fukazawa K, Sawada K, Koike T, Kuwabara M, Ikeda H, Ikebuchi K (2002) Ex vivo expansion of human UC blood primitive hematopoietic progenitors and transplantable stem cells using human primary BM stromal cells and human AB serum. Cytotherapy 4:109–118

  159. Yin Y, Lim YK, Salto-Tellez M, Ng SC, Lin CS, Lim SK (2002) AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo. Stem Cells 20:338–346

  160. Ying QL, Nichols J, Evans EP, Smith AG (2002) Changing potency by spontaneous fusion. Nature 416:545–548

  161. Yoshikawa T, Peel SA, Gladstone JR, Davies JE (1997) Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation. Biomed Mater Eng 7:369–377

  162. Yoshikawa Y, Hirayama F, Kanai M, Nakajo S, Ohkawara J, Fujihara M, Yamaguchi M, Sato N, Kasai M, Sekiguchi S, Ikebuchi K (2000) Stromal cell-independent differentiation of human cord blood CD34+CD38− lymphohematopoietic progenitors toward B cell lineage. Leukemia 14:727–734

  163. Yuan HP, Ge J, Duan YH, Wang YM, Wang LN, Yang BB (2003) Induced differentiation of neural stem cells from subependymal zone into retinal cells in vitro. Zhonghua Yan Ke Za Zhi 39:357–360

  164. Zhang WL, Tsuneishi S, Nakamura H (2001) Induction of heat shock proteins and its effects on glial differentiation in rat C6 glioblastoma cells. Kobe J Med Sci 47:77–95

Download references

Author information

Correspondence to Soon Chye Ng.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Heng, B.C., Cao, T., Haider, H.K. et al. An overview and synopsis of techniques for directing stem cell differentiation in vitro. Cell Tissue Res 315, 291–303 (2004). https://doi.org/10.1007/s00441-003-0847-5

Download citation


  • Stem cells
  • Plasticity
  • In vitro
  • Differentiation
  • Transplantation