成人骨髓间充质干细胞体外培养及神经元样细胞定向诱导分化的研究
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摘要
目的:研究成人骨髓间充质干细胞(hMSC)的生物特性和发生,发育的规律,探讨其在临床应用,体外扩增的过程中的安全性,以及在临床应用的过程中是否会发生恶性转化。
方法:我们研究了体外培养的hMSC,在不同的培养时间点,发生恶性转化的敏感性。穿刺抽取10名健康志愿者的骨髓,分离提取得到hMSC,体外培养至衰老或需要传代(P) 25,8周至12周(hMSC的衰老期),干预终止之前,密切观察监视hMSC的变化。通过阵列比较基因组杂交,常规核型分析和端粒荧光原位杂交,研究分析长期培养之前和之后干细胞的遗传特性。检测分析不同传代次数和阶段,hMSC端粒酶的表达活性,端粒酶逆转录酶(hTERT)的转录物,和端粒延长的具体机制。
结果:不同的志愿者之间,hMSC的增殖能力,和寿命有着巨大的变化。其中八个志愿者,hMSC增殖能力在衰老之前逐渐减弱。余下的两个志愿者,为了进行数据分析在P25阶段,hMSC的培养被打断。阵列比较基因组杂交阵列全息图和细胞遗传学分析显示,干细胞在体外培养扩增没有出现染色体异常;端粒酶活性未变,hTERT没有转录表达,端粒在培养阶段被缩短。端粒的延长机制没有发现和证实。
结论:骨髓来源的干细胞可以在体外安全地扩增,不易发生恶性转化,从而使hMSC的更加适合于干细胞疗法的临床应用。
OBJECTIVE: Significant improvement in the understanding of mesenchymal stem cell (HMSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that HMSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)–derived HMSCs at different in vitro culture time points.
METHODS: HMSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. HMSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of HMSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping,and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. HMSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages.
Results: A huge variability in terms of proliferative capacity and HMSCs life span was noted between donors. In eight of 10 donors, HMSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two HMSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that HMSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the HMSCs tested.
Conclusion: BM-derived HMSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.
方法:我们研究了体外培养的hMSC,在不同的培养时间点,发生恶性转化的敏感性。穿刺抽取10名健康志愿者的骨髓,分离提取得到hMSC,体外培养至衰老或需要传代(P) 25,8周至12周(hMSC的衰老期),干预终止之前,密切观察监视hMSC的变化。通过阵列比较基因组杂交,常规核型分析和端粒荧光原位杂交,研究分析长期培养之前和之后干细胞的遗传特性。检测分析不同传代次数和阶段,hMSC端粒酶的表达活性,端粒酶逆转录酶(hTERT)的转录物,和端粒延长的具体机制。
结果:不同的志愿者之间,hMSC的增殖能力,和寿命有着巨大的变化。其中八个志愿者,hMSC增殖能力在衰老之前逐渐减弱。余下的两个志愿者,为了进行数据分析在P25阶段,hMSC的培养被打断。阵列比较基因组杂交阵列全息图和细胞遗传学分析显示,干细胞在体外培养扩增没有出现染色体异常;端粒酶活性未变,hTERT没有转录表达,端粒在培养阶段被缩短。端粒的延长机制没有发现和证实。
结论:骨髓来源的干细胞可以在体外安全地扩增,不易发生恶性转化,从而使hMSC的更加适合于干细胞疗法的临床应用。
OBJECTIVE: Significant improvement in the understanding of mesenchymal stem cell (HMSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that HMSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)–derived HMSCs at different in vitro culture time points.
METHODS: HMSCs were isolated from BM of 10 healthy donors and propagated in vitro until reaching either senescence or passage (P) 25. HMSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of HMSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping,and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. HMSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages.
Results: A huge variability in terms of proliferative capacity and HMSCs life span was noted between donors. In eight of 10 donors, HMSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two HMSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that HMSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the HMSCs tested.
Conclusion: BM-derived HMSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches.
引文
[1] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418(6893):41–49.
[2] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3(4):393–403.
[3] Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9(5): 641–650.
[4] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276(5309):71–74.
[5] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147.
[6] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–317.
[7] Tao H, Rao R, Ma DD. Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition. Dev Growth Differ. 2005;47(6):423–433.
[8] Long X, Olszewski M, Huang W, Kletzel M. Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells. Stem Cells Devel. 2005;14(1):65–69.
[9] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000;164(2):247–256.
[10] Mahmood A, Lu D, Yi L, Chen JL, Chopp M. Intracranial bone marrow transplantation after traumatic brain injury improving functional outcome in adult rats. J Neurosurg. 2001;94(4): 589–595.
[11] Mahmood A, Lu D, Lu M, Chopp M. Treatment of traumatic brain injury in adult rats with intravenous administration of human bone marrow stromal cells. Neurosurgery. 2003;53(3):697–702; discussion-3.
[12] Reynolds BA,Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707–1710.
[13] McKay R. Stem cells in the central nervous system. Science. 1997;276(5309):66–71.
[14] McKay R, Renfranz P, Cunningham M. Immortalized stem cells from the central nervous system. Comptes rendus de l’Academie des sciences. 1993;316(12):1452–1457.
[15] Gage FH. Mammalian neural stem cells. Science. 2000; 287(5457):1433–1438.
[16] Okano H. Stem cell biology of the central nervous system. J Neurosci Res. 2002;69(6):698–707.
[17] Murayama A, Matsuzaki Y, Kawaguchi A, Shimazaki T, Okano H. Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J Neurosci Res. 2002;69(6):837–847.
[18] Xu Y, Kimura K, Matsumoto N, Ide C. Isolation of neural stem cells from the forebrain of deceased early postnatal and adult rats with protracted post-mortem intervals. J Neurosci Res. 2003;74(4):533–540.
[19] Secco M, Zucconi E, Vieira NM, et al. Multipotent stem cells from umbilical cord: cord is richer than blood! Stem Cells. 2007.
[20] Yasuhara T, Matsukawa N, Yu G, et al. Transplantation of cryopreserved human bone
[21] marrow-derived multipotent adult progenitor cells for neonatal hypoxic-ischemic injury: targeting the hippocampus. Rev. Neurosci. 2006;17(1–2):215–225.
[22] Tang Y, Yasuhara T, Hara K, et al. Transplantation of bone marrow-derived stem cells: a promising therapy for stroke. Cell Transplant. 2007;16(2):159–169.
[23] Consensus conference. Rehabilitation of persons with traumatic brain injury. NIH Consensus Development Panel on Rehabilitation of Persons With Traumatic Brain Injury. JAMA. 1999;282(10):974–983.
[24] Mahmood A, Lu D, Wang L, Li Y, Lu M, Chopp M. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery. 2001;49(5):1196–203; discussion 203–204.
[25] Lu D, Mahmood A, Wang L, Li Y, Lu M, Chopp M. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport. 2001;12(3):559–563.
[26] Lu D, Li Y,Wang L, Chen J, Mahmood A, Chopp M. Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma. 2001;18(8):813–819.
[27] Tolar J, O’Shaughnessy MJ, Panoskaltsis-Mortari A, et al. Host factors that impact the biodistribution and persistence of multipotent adult progenitor cells. Blood. 2006;107(10): 4182–4188.
[28] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Long-term recovery after bone marrow stromal cell treatment of traumatic brain injury in rats. J Neurosurg. 2006;104(2):272–277.
[29] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Treatment of traumatic brain injury with a combination therapy of marrow
[30] stromal cells and atorvastatin in rats. Neurosurgery. 2007;60(3): 546–53; discussion 53–54.
[31] Lu D, Mahmood A, Qu C, Hong X, Kaplan D, Chopp M. Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury. Neurosurgery. 2007;61(3):596–602; discussion-3.
[32] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Human marrow stromal cell treatment provides long-lasting benefit after traumatic brain injury in rats. Neurosurgery. 2005;57(5):1026–1031; discussion 1031.
[33] Sinson G, Voddi M, McIntosh TK. Combined fetal neural transplantation and nerve growth factor infusion: effects on neurological outcome following fluid-percussion brain injury in the rat. J Neurosurg. 1996;84(4):655–662.
[34] Philips MF, Muir JK, Saatman KE, et al. Survival and integration of transplanted postmitotic human neurons following experimental brain injury in immunocompetent rats. J Neurosurg. 1999;90(1):116–124.
[35] Muir JK, Raghupathi R, Saatman KE, et al. Terminally differentiated human neurons survive and integrate following transplantation into the traumatically injured rat brain. J Neurotrauma. 1999;16(5):403–414.
[36] Zhang C, Saatman KE, Royo NC, et al. Delayed transplantation of human neurons following brain injury in rats: a long-term graft survival and behavior study. J Neurotrauma. 2005;22(12): 1456–1474.
[37] Gao J, Prough DS, McAdoo DJ, et al. Transplantation of primed human fetal neural stem cells improves cognitive function in rats after traumatic brain injury. Exp Neurol. 2006;201(2):281–292.
[38] Englund U, Bjorklund A, Wictorin K. Migration patterns and phenotypic differentiation of long-term expanded human neural progenitor cells after transplantation into the adult rat brain. Brain Res Dev Brain Res. 2002;134(1–2):123–141.
[39] Englund U, Fricker-Gates RA, Lundberg C, Bjorklund A, Wictorin K. Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections. Exp Neurol. 2002;173(1):1–21.
[40] Vescovi AL, Gritti A, Galli R, Parati EA. Isolation and intracerebral grafting of
[41] nontransformed multipotential embryonic human CNS stem cells. J Neurotrauma. 1999;16(8):689–693.
[42] Englund U, Bjorklund A, Wictorin K, Lindvall O, Kokaia M. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci U S A. 2002;99(26):17089–94.
[43] Hagan M, Wennersten A, Meijer X, Holmin S, Wahlberg L, Mathiesen T. Neuroprotection by human neural progenitor cells after experimental contusion in rats. Neurosci Lett. 2003;351(3): 149–152.
[44] Wennersten A, Holmin S, Al Nimer F, Meijer X, Wahlberg LU, Mathiesen T. Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression. Exp Neurol. 2006;199(2):339–347.
[45] Al Nimer F, Wennersten A, Holmin S, Meijer X, Wahlberg L, Mathiesen T. MHC expression after human neural stem cell transplantation to brain contused rats. Neuroreport. 2004;15(12): 1871–1875.
[46] Shear DA, Tate MC, Archer DR, et al. Neural progenitor cell transplants promote long-term functional recovery after traumatic brain injury. Brain Res. 2004;1026(1):11–22.
[47] Tate MC, Shear DA, Hoffman SW, Stein DG, Archer DR, LaPlaca MC. Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain. Cell Transplant. 2002;11(3):283–295.
[48] Renfranz PJ, Cunningham MG, McKay RD. Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell. 1991;66(4):713–729.
[49] Sinden JD, Rashid-Doubell F, Kershaw TR, et al. Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischaemia-lesioned hippocampus. Neuroscience. 1997;81(3):599–608.
[50] Ryder EF, Snyder EY, Cepko CL. Establishment and characterization of multipotent neural cell lines using retrovirus vectormediated oncogene transfer. J Neurobiol. 1990;21(2):356–375.
[51] Shindo T, Matsumoto Y, Wang Q, Kawai N, Tamiya T, Nagao S. Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury. J Med Invest. 2006;53(1–2):42–51.
[52] Lu D, Sanberg PR, Mahmood A, et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant. 2002;11(3): 275–281.
[53] Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet. 2006;367(9524):1747–1757.
[54] Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century.Lancet Neurol. 2003;2(1):43–53.
[55] van der Worp HB, van Gijn J. Clinical practice. Acute ischemic stroke. New Engl J Med. 2007;357(6):572–579.
[56] Li Y, Chen J, Chopp M. Adult bone marrow transplantation after stroke in adult rats. Cell Transplant. 2001;10(1):31–40.
[57] Li Y, Chopp M, Chen J, et al. Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab. 2000;20(9):1311–1319.
[58] Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Experimental Neurol. 2002;174(1): 11–20.
[59] Li Y, Chen J, Wang L, Lu M, Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology. 2001;56(12):1666–1672.
[60] Shen LH, Li Y, Chen J, et al. Intracarotid transplantation of bone marrow stromal cells increases axon-myelin remodeling after stroke. Neuroscience. 2006;137(2):393–399.
[61] Chu K, Kim M, Park KI, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ischemia. Brain Res. 2004;1016(2):145–153.
[62] Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia. Neurosci Lett. 2003;343(2):129–133.
[63] Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke. 2003;34(9):2258–2263.
[64] Ishibashi S, Sakaguchi M, Kuroiwa T, et al. Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils. J Neurosci Res. 2004;78(2):215–223.
[65] Kelly S, Bliss TM, Shah AK, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci U S A. 2004;101(32): 11839–44.
[66] Mi R, Luo Y, Cai J, Limke TL, Rao MS, Hoke A. Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors. Exp Neurol. 2005;194(2):301–319.
[67] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement.Cytotherapy 2006;8:315–317.
[68] Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical use. Exp Hemat 2000;28: 875–884.
[69] Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568–584.
[70] Jorgensen C, Gordeladze J, Noel D. Tissue engineering through autologous mesenchymal stem cells. Curr Opin Biotechnol 2004;15:406–410.
[71] Koc ON, Gerson SL, Cooper BM, et al. Rapid hematopoietic recovery after co-infusion of autologous-blood stem cells and culture-expanded marrow mesenchymalstem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–316.
[72] Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transpl 2005;11:389–398.
[73] Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363:1439–41.
[74] Frassoni F, Labopin M, Bacigalupo A, et al. Expanded mesenchymal stem cells (HMSC), co-infused with HLAidentical hematopoietic stem cell transplant, reduce acute and chronic graft versus host disease: a matched pair analysis [abstract]. Bone Marrow Transplant 2002; 29:75.
[75] Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
[76] Le Blanc K, Gotherstrom C, Ringden O, et al. Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation 2005;79:1607–614.
[77] Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy and Hurler syndrome. Bone Marrow Transplant 2002;30:215–222.
[78] Puontos I, Jones E, Tzioupis C, McGonagle D, Giannoudis PV. Growing bone and cartilage. Role of mesenchymal stem cells. J Bone Joint Surg Br 2006;88:
[79] 426.
[80] Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385–6.
[81] Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:429–435.
[82] Prockop DJ. Marrow stromal cells as a stem cells from nonhematopoietic tissues. Science 1997;276:71–74.
[83] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
[84] Bianco P, Gehron Robey P. Marrow stromal stem cells. J Clin Invest 2000;105:1663–8.
[85] Im G-I, Shin Y-W, Lee K-B. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage 2005;13:845–853.
[86] in’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338–45.
[87] Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035–3039.
[88] Miura M, Miura Y, Padilla-Nash HM, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095–1103.
[89] Houghton JM, Stoicov C, Nomura S, et al. Gastric cancer originating from bone marrow-derived cells. Science 2004;306:1568–71.
[90] in’t Anker PS, Noort WA, Scherjon SA, Kleijburgvan der Keur C, Kruisselbrink AB, van Bezooijen RL. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003;88: 845–852.
[91] Bernardo ME, Avanzini MA, Perotti C, et al. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J Cell Physiol 2007;211:121–130.
[92] Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–2015.
[93] Zaffaroni N, Villa R, Pastorino U, et al. Lack of telomerase activity in lung carcinoids is dependent on human telomerase reverse transcriptase transcription and alternative splicing and is associated with long telomeres. Clin Cancer Res 2005;11:2832–2839.
[94] Henson JD, Hannay JA, McCarthy SW, et al. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res 2005;11:217–225.
[95] Villa R, Folini M, Perego P, et al. Telomerase activity and telomere length in human ovariancancer and melanoma cell lines: correlation with sensitivity to DNA damaging agents. Int J Oncol 2000;16:995–1002.
[96] Grant JD, Broccoli D, Muquit M, Manion FJ, Tisdall J, Ochs MF. Telometric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels. BioTechniques 2001;31: 1314–1318.
[97] Das P, Kotilingam D, Kochin B, et al. High prevalence of p53 exon 4 mutations in soft-tissue sarcoma. Cancer 2007;109:2323–2333.
[98] Wang Y, Huso DL, Harrington J, et al. Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 2005;7:509–519.
[99] Caterson EJ, Nesti LJ, Danielson KJ, Tuan RS. Human marrow derived mesenchymal progenitor cells: isolation culture, expansion and analysis of differentiation. Mol Biotechnol 2002;20:245–256.
[100] Harrington L, Zhou W, McPhail T, et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev 1997;11:3109–3115.
[101] Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res 1998;58:4168–4172.
[102] Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med 1997;3:1271–1274.
[103] Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene 2002;21:598–610.
[2] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3(4):393–403.
[3] Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9(5): 641–650.
[4] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276(5309):71–74.
[5] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147.
[6] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–317.
[7] Tao H, Rao R, Ma DD. Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition. Dev Growth Differ. 2005;47(6):423–433.
[8] Long X, Olszewski M, Huang W, Kletzel M. Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells. Stem Cells Devel. 2005;14(1):65–69.
[9] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000;164(2):247–256.
[10] Mahmood A, Lu D, Yi L, Chen JL, Chopp M. Intracranial bone marrow transplantation after traumatic brain injury improving functional outcome in adult rats. J Neurosurg. 2001;94(4): 589–595.
[11] Mahmood A, Lu D, Lu M, Chopp M. Treatment of traumatic brain injury in adult rats with intravenous administration of human bone marrow stromal cells. Neurosurgery. 2003;53(3):697–702; discussion-3.
[12] Reynolds BA,Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707–1710.
[13] McKay R. Stem cells in the central nervous system. Science. 1997;276(5309):66–71.
[14] McKay R, Renfranz P, Cunningham M. Immortalized stem cells from the central nervous system. Comptes rendus de l’Academie des sciences. 1993;316(12):1452–1457.
[15] Gage FH. Mammalian neural stem cells. Science. 2000; 287(5457):1433–1438.
[16] Okano H. Stem cell biology of the central nervous system. J Neurosci Res. 2002;69(6):698–707.
[17] Murayama A, Matsuzaki Y, Kawaguchi A, Shimazaki T, Okano H. Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J Neurosci Res. 2002;69(6):837–847.
[18] Xu Y, Kimura K, Matsumoto N, Ide C. Isolation of neural stem cells from the forebrain of deceased early postnatal and adult rats with protracted post-mortem intervals. J Neurosci Res. 2003;74(4):533–540.
[19] Secco M, Zucconi E, Vieira NM, et al. Multipotent stem cells from umbilical cord: cord is richer than blood! Stem Cells. 2007.
[20] Yasuhara T, Matsukawa N, Yu G, et al. Transplantation of cryopreserved human bone
[21] marrow-derived multipotent adult progenitor cells for neonatal hypoxic-ischemic injury: targeting the hippocampus. Rev. Neurosci. 2006;17(1–2):215–225.
[22] Tang Y, Yasuhara T, Hara K, et al. Transplantation of bone marrow-derived stem cells: a promising therapy for stroke. Cell Transplant. 2007;16(2):159–169.
[23] Consensus conference. Rehabilitation of persons with traumatic brain injury. NIH Consensus Development Panel on Rehabilitation of Persons With Traumatic Brain Injury. JAMA. 1999;282(10):974–983.
[24] Mahmood A, Lu D, Wang L, Li Y, Lu M, Chopp M. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery. 2001;49(5):1196–203; discussion 203–204.
[25] Lu D, Mahmood A, Wang L, Li Y, Lu M, Chopp M. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport. 2001;12(3):559–563.
[26] Lu D, Li Y,Wang L, Chen J, Mahmood A, Chopp M. Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury. J Neurotrauma. 2001;18(8):813–819.
[27] Tolar J, O’Shaughnessy MJ, Panoskaltsis-Mortari A, et al. Host factors that impact the biodistribution and persistence of multipotent adult progenitor cells. Blood. 2006;107(10): 4182–4188.
[28] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Long-term recovery after bone marrow stromal cell treatment of traumatic brain injury in rats. J Neurosurg. 2006;104(2):272–277.
[29] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Treatment of traumatic brain injury with a combination therapy of marrow
[30] stromal cells and atorvastatin in rats. Neurosurgery. 2007;60(3): 546–53; discussion 53–54.
[31] Lu D, Mahmood A, Qu C, Hong X, Kaplan D, Chopp M. Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury. Neurosurgery. 2007;61(3):596–602; discussion-3.
[32] Mahmood A, Lu D, Qu C, Goussev A, Chopp M. Human marrow stromal cell treatment provides long-lasting benefit after traumatic brain injury in rats. Neurosurgery. 2005;57(5):1026–1031; discussion 1031.
[33] Sinson G, Voddi M, McIntosh TK. Combined fetal neural transplantation and nerve growth factor infusion: effects on neurological outcome following fluid-percussion brain injury in the rat. J Neurosurg. 1996;84(4):655–662.
[34] Philips MF, Muir JK, Saatman KE, et al. Survival and integration of transplanted postmitotic human neurons following experimental brain injury in immunocompetent rats. J Neurosurg. 1999;90(1):116–124.
[35] Muir JK, Raghupathi R, Saatman KE, et al. Terminally differentiated human neurons survive and integrate following transplantation into the traumatically injured rat brain. J Neurotrauma. 1999;16(5):403–414.
[36] Zhang C, Saatman KE, Royo NC, et al. Delayed transplantation of human neurons following brain injury in rats: a long-term graft survival and behavior study. J Neurotrauma. 2005;22(12): 1456–1474.
[37] Gao J, Prough DS, McAdoo DJ, et al. Transplantation of primed human fetal neural stem cells improves cognitive function in rats after traumatic brain injury. Exp Neurol. 2006;201(2):281–292.
[38] Englund U, Bjorklund A, Wictorin K. Migration patterns and phenotypic differentiation of long-term expanded human neural progenitor cells after transplantation into the adult rat brain. Brain Res Dev Brain Res. 2002;134(1–2):123–141.
[39] Englund U, Fricker-Gates RA, Lundberg C, Bjorklund A, Wictorin K. Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections. Exp Neurol. 2002;173(1):1–21.
[40] Vescovi AL, Gritti A, Galli R, Parati EA. Isolation and intracerebral grafting of
[41] nontransformed multipotential embryonic human CNS stem cells. J Neurotrauma. 1999;16(8):689–693.
[42] Englund U, Bjorklund A, Wictorin K, Lindvall O, Kokaia M. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci U S A. 2002;99(26):17089–94.
[43] Hagan M, Wennersten A, Meijer X, Holmin S, Wahlberg L, Mathiesen T. Neuroprotection by human neural progenitor cells after experimental contusion in rats. Neurosci Lett. 2003;351(3): 149–152.
[44] Wennersten A, Holmin S, Al Nimer F, Meijer X, Wahlberg LU, Mathiesen T. Sustained survival of xenografted human neural stem/progenitor cells in experimental brain trauma despite discontinuation of immunosuppression. Exp Neurol. 2006;199(2):339–347.
[45] Al Nimer F, Wennersten A, Holmin S, Meijer X, Wahlberg L, Mathiesen T. MHC expression after human neural stem cell transplantation to brain contused rats. Neuroreport. 2004;15(12): 1871–1875.
[46] Shear DA, Tate MC, Archer DR, et al. Neural progenitor cell transplants promote long-term functional recovery after traumatic brain injury. Brain Res. 2004;1026(1):11–22.
[47] Tate MC, Shear DA, Hoffman SW, Stein DG, Archer DR, LaPlaca MC. Fibronectin promotes survival and migration of primary neural stem cells transplanted into the traumatically injured mouse brain. Cell Transplant. 2002;11(3):283–295.
[48] Renfranz PJ, Cunningham MG, McKay RD. Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell. 1991;66(4):713–729.
[49] Sinden JD, Rashid-Doubell F, Kershaw TR, et al. Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischaemia-lesioned hippocampus. Neuroscience. 1997;81(3):599–608.
[50] Ryder EF, Snyder EY, Cepko CL. Establishment and characterization of multipotent neural cell lines using retrovirus vectormediated oncogene transfer. J Neurobiol. 1990;21(2):356–375.
[51] Shindo T, Matsumoto Y, Wang Q, Kawai N, Tamiya T, Nagao S. Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury. J Med Invest. 2006;53(1–2):42–51.
[52] Lu D, Sanberg PR, Mahmood A, et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant. 2002;11(3): 275–281.
[53] Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet. 2006;367(9524):1747–1757.
[54] Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century.Lancet Neurol. 2003;2(1):43–53.
[55] van der Worp HB, van Gijn J. Clinical practice. Acute ischemic stroke. New Engl J Med. 2007;357(6):572–579.
[56] Li Y, Chen J, Chopp M. Adult bone marrow transplantation after stroke in adult rats. Cell Transplant. 2001;10(1):31–40.
[57] Li Y, Chopp M, Chen J, et al. Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab. 2000;20(9):1311–1319.
[58] Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Experimental Neurol. 2002;174(1): 11–20.
[59] Li Y, Chen J, Wang L, Lu M, Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology. 2001;56(12):1666–1672.
[60] Shen LH, Li Y, Chen J, et al. Intracarotid transplantation of bone marrow stromal cells increases axon-myelin remodeling after stroke. Neuroscience. 2006;137(2):393–399.
[61] Chu K, Kim M, Park KI, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ischemia. Brain Res. 2004;1016(2):145–153.
[62] Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia. Neurosci Lett. 2003;343(2):129–133.
[63] Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke. 2003;34(9):2258–2263.
[64] Ishibashi S, Sakaguchi M, Kuroiwa T, et al. Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils. J Neurosci Res. 2004;78(2):215–223.
[65] Kelly S, Bliss TM, Shah AK, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci U S A. 2004;101(32): 11839–44.
[66] Mi R, Luo Y, Cai J, Limke TL, Rao MS, Hoke A. Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors. Exp Neurol. 2005;194(2):301–319.
[67] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement.Cytotherapy 2006;8:315–317.
[68] Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical use. Exp Hemat 2000;28: 875–884.
[69] Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004;36:568–584.
[70] Jorgensen C, Gordeladze J, Noel D. Tissue engineering through autologous mesenchymal stem cells. Curr Opin Biotechnol 2004;15:406–410.
[71] Koc ON, Gerson SL, Cooper BM, et al. Rapid hematopoietic recovery after co-infusion of autologous-blood stem cells and culture-expanded marrow mesenchymalstem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–316.
[72] Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transpl 2005;11:389–398.
[73] Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363:1439–41.
[74] Frassoni F, Labopin M, Bacigalupo A, et al. Expanded mesenchymal stem cells (HMSC), co-infused with HLAidentical hematopoietic stem cell transplant, reduce acute and chronic graft versus host disease: a matched pair analysis [abstract]. Bone Marrow Transplant 2002; 29:75.
[75] Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.
[76] Le Blanc K, Gotherstrom C, Ringden O, et al. Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation 2005;79:1607–614.
[77] Koc ON, Day J, Nieder M, Gerson SL, Lazarus HM, Krivit W. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy and Hurler syndrome. Bone Marrow Transplant 2002;30:215–222.
[78] Puontos I, Jones E, Tzioupis C, McGonagle D, Giannoudis PV. Growing bone and cartilage. Role of mesenchymal stem cells. J Bone Joint Surg Br 2006;88:
[79] 426.
[80] Quarto R, Mastrogiacomo M, Cancedda R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 2001;344:385–6.
[81] Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:429–435.
[82] Prockop DJ. Marrow stromal cells as a stem cells from nonhematopoietic tissues. Science 1997;276:71–74.
[83] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
[84] Bianco P, Gehron Robey P. Marrow stromal stem cells. J Clin Invest 2000;105:1663–8.
[85] Im G-I, Shin Y-W, Lee K-B. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage 2005;13:845–853.
[86] in’t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338–45.
[87] Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005;65:3035–3039.
[88] Miura M, Miura Y, Padilla-Nash HM, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095–1103.
[89] Houghton JM, Stoicov C, Nomura S, et al. Gastric cancer originating from bone marrow-derived cells. Science 2004;306:1568–71.
[90] in’t Anker PS, Noort WA, Scherjon SA, Kleijburgvan der Keur C, Kruisselbrink AB, van Bezooijen RL. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003;88: 845–852.
[91] Bernardo ME, Avanzini MA, Perotti C, et al. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J Cell Physiol 2007;211:121–130.
[92] Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–2015.
[93] Zaffaroni N, Villa R, Pastorino U, et al. Lack of telomerase activity in lung carcinoids is dependent on human telomerase reverse transcriptase transcription and alternative splicing and is associated with long telomeres. Clin Cancer Res 2005;11:2832–2839.
[94] Henson JD, Hannay JA, McCarthy SW, et al. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clin Cancer Res 2005;11:217–225.
[95] Villa R, Folini M, Perego P, et al. Telomerase activity and telomere length in human ovariancancer and melanoma cell lines: correlation with sensitivity to DNA damaging agents. Int J Oncol 2000;16:995–1002.
[96] Grant JD, Broccoli D, Muquit M, Manion FJ, Tisdall J, Ochs MF. Telometric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels. BioTechniques 2001;31: 1314–1318.
[97] Das P, Kotilingam D, Kochin B, et al. High prevalence of p53 exon 4 mutations in soft-tissue sarcoma. Cancer 2007;109:2323–2333.
[98] Wang Y, Huso DL, Harrington J, et al. Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 2005;7:509–519.
[99] Caterson EJ, Nesti LJ, Danielson KJ, Tuan RS. Human marrow derived mesenchymal progenitor cells: isolation culture, expansion and analysis of differentiation. Mol Biotechnol 2002;20:245–256.
[100] Harrington L, Zhou W, McPhail T, et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev 1997;11:3109–3115.
[101] Ulaner GA, Hu JF, Vu TH, Giudice LC, Hoffman AR. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res 1998;58:4168–4172.
[102] Bryan TM, Englezou A, Dalla-Pozza L, Dunham MA, Reddel RR. Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat Med 1997;3:1271–1274.
[103] Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene 2002;21:598–610.