- Email: [email protected]
Adult Rat and Human Bone Marrow Stromal Stem Cells Differentiate into Neurons
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
Black and Woodbury
Adult Rat and Human Bone Marrow Stromal Stem Cells Differentiate into Neurons Ira B. Black1 and Dale Woodbury1 Submitted 05/02/01 (Communicated by M. Lichtman, M.D., 05/05/01)
SUMMARY OVERVIEW
INTRODUCTION
Characterization of Stromal Cells Stem cells have been detected in many adult tissues, participating in normal replacement and repair, while undergoing self-renewal (1–11). A subset of bone marrow stem cells constitutes one subtype, capable of differentiating into bone, cartilage, muscle, tendon, fat, and other mesenchymal lineages in vitro (2, 4 –9). They have been termed marrow stromal cells (MSCs). The recent detection of stem cell populations in the central nervous system (CNS) has attracted great attention, since the brain has long been regarded as incapable of regrowth (12, 13). Neural stem cells (NSCs) undergo expansion and differentiate into neurons, astrocytes, and oligodendrocytes in vitro (12, 14 –16). NSCs transplanted into the adult rodent brain survive and differentiate into neurons and glia, raising the possibility of new therapeutic approaches (17–22). However, the inaccessibility of NSCs deep in the brain severely limits clinical utility. A recent report demonstrating that NSCs generate hematopoietic cells in vivo suggests that stem cell populations may be less restricted than was previously believed (23). Evidence that MSCs introduced into the lateral ventricles of neonatal mice can differentiate into astrocytes and neurofilament-containing cells lends support to this contention (24). The present precis briefly describes the differentiation of rat and human MSCs into neurons, and the potential advantages of this approach in the treatment of neurologic disease (25).
Rat mesenchymal stromal cells (rMSCs) were isolated from the femurs of adult rats and propagated in vitro (26). Fluorescent cell sorting at passage 1 demonstrated that the cells were negative for CD11b and CD45, surface markers associated with lymphohematopoietic cells. Consequently, there was no evidence of hematopoietic precursors in the cultures. In contrast, the rMSCs did express CD90, consistent with their undifferentiated state. Initially, with neuronal differentiation, untreated rMSCs were further characterized by staining positively for CD44 and CD71, consistent with previous reports (9, 27). Differentiation into Neurons To induce the neuronal phenotype, rMSCs were maintained in subconfluent cultures in serum-containing medium supplemented with 1 mM -mercaptoethanol (BME) for 24 h (25). To induce neuronal differentiation, the cells were transferred to serum-free medium containing 1–10 mM BME (SFM/BME). Within 60 min of exposure to SFM/BME, changes in morphology of some of the rMSCs were apparent. Responsive cells progressively assumed neuronal morphological traits over the first 3 h. Initially, cytoplasm in the flat rMSCs retracted toward the nucleus, forming a contracted multipolar, cell body, leaving membranous, process-like extensions peripherally
Correspondence and reprint requests to: Ira B. Black. Fax: (732)235-5885. E-mail: [email protected]. 1 Department of Neuroscience and Cell Biology, UMDNJ–Robert Wood Johnson Medical School, 675 Hoes Lane, CABM 342, Piscataway, New Jersey 08854. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
632
Black and Woodbury
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
(0 –90 min). Cells exhibited increased expression of the neuronal marker NSE within 30 min of treatment. Over the subsequent 2 h, cell bodies became increasingly spherical and refractile, exhibiting a typical neuronal perikaryal appearance. Processes continued to elaborate, displaying primary and secondary branches, growth cone-like terminal expansions and putative filopodial extensions. To characterize neuronal differentiation further, we fixed BME-treated cultures after 5 h and stained them for the neuronal marker NSE (25). Unresponsive, flat rMSCs expressed very low, but detectable, levels of NSE protein, consistent with previous detection of minute amounts of protein and/or message in cells of bone marrow origin (28 –30). Progressive transition of rMSCs to a neuronal phenotype coincided with increased expression of NSE. rMSC-derived neurons displayed distinct neuronal morphologies, ranging from simple bipolar to large, extensively branched multipolar cells. Clusters of differentiated cells exhibited intense NSE positivity, and processes formed extensive networks. Western blot analysis confirmed the expression of low levels of NSE protein in uninduced rMSCs. Induction of the neuronal phenotype dramatically increased NSE expression, consistent with the immunocytochemical data. To characterize neuronal identity further, MSCs treated with DMSO/BHA (butylated hydroxyanisole, an antioxidant) were stained for neurofilament-M (NF-M), a neuron-specific intermediate filament that helps initiate neurite elongation (31). We previously found that BME treatment of MSCs elicited increased expression of NF-M in cells exhibiting neuronal morphologies. Most cells displaying rounded cell bodies with processes after DMSO/BHA exposure expressed high levels of NF-M, whereas flat, undifferentiated cells did not. Preadsorption of NF-M antibody with purified NF-M protein abolished staining, establishing specificity (25). We examined DMSO/BHA-treated cultures for the presence of tau, a neuron-specific microtubule-associated protein expressed by differentiating neurons (32). Cells exhibiting a neuronal morphology expressed tau protein in the cell body
as well as in the processes, whereas undifferentiated flat cells were tau-negative (25). To investigate neuronal characteristics further, we examined differentiated cultures for NeuN, a neuron-specific marker expressed in postmitotic cells (33). A subset of cells exhibiting rounded cell bodies and processes stained for NeuN expression, whereas neighboring cells exhibiting distinct neuronal morphologies were NeuN-negative. This observation suggests that a subset of NSE-positive cells are postmitotic neurons. Quantitation of Neuronal Differentiation With this induction protocol, a variable number of cells underwent neuronal differentiation, although the response generally exceeded 50%. To optimize differentiation, we modified the preinduction protocol. Addition of bFGF (10 ng/ml) and elimination of BME from the preinduction medium increased the proportion of cells exhibiting neuronal traits, and the response was more consistent within and between experiments. To quantitate this response, rMSCs were treated with the modified preinduction protocol, and induced to differentiate with DMSO/BHA. Cells were fixed after 5 h, stained for the neuronal markers NSE and NF-M, and the percentage of neuronal cells was determined. The majority of rMSCs treated in this manner exhibited neuronal morphologies and stained positively for NSE (78.2 ⫾ 2.3%) and NF-M (79.2% ⫾ 2.5%) expression (25). Long-Term Differentiation A dramatic aspect of rMSC neuronal differentiation is the rapidity of the response. As a result, our analysis focused on changes that occurred within the first 5 h of differentiation. However, long-term differentiation of these cells will be important to our understanding of this process. To address this issue, we monitored expression of the nestin gene product in rMSC-derived neurons at 5 h, 1 day, and 6 days postdifferentiation. Nestin, an intermediate filament protein, is expressed in neuroepithelial neuronal precursor 633
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
stem cells, and its expression decreases with neuronal maturation (34). A subset of rMSC-derived neurons expressed high levels of nestin protein at 5 h, and the proportion of nestin-positive cells decreased with time. By 6 days postinduction, there was no detectable nestin expression in any rMSC-derived neurons, consistent with ongoing maturation with time (25). We compared the time course of expression of the transitional trait, nestin, to that of a mature phenotypic character, trkA, the biologically active nerve growth factor receptor. TrkA was detectable at 5 h, the earliest time examined and, in contrast to nestin, persisted unchanged through 6 days (25).
Black and Woodbury
similar to that observed for rMSCs. Contracted cell bodies elaborated processes and stained intensely for NSE expression within 3 h. Transitional cells were also evident. Many processes elaborated by hMSC-derived neurons exhibited terminal bulbs, which may represent growth cones. These cells also expressed NF-M, consistent with neuronal differentiation. PERSPECTIVE Our observations suggest that intrinsic genomic mechanisms of commitment, lineage restriction, and cell fate are plastic. The environment apparently can evoke the expression of pluripotentiality that far exceeds traditional fate restrictions of cells derived from the classical embryonic germ layers. The adult cells are both self-renewing and pluripotential (2–9), thereby satisfying many of the criteria of a stem cell population. The use of MSCs to generate neurons for transplantation confers a number of potential therapeutic advantages. Autologous transplantation eliminates the hazards of immunorejection, and obviates the need for toxic immunosuppressive agents. The bone marrow is a safe, accessible source, overcoming the risks of obtaining stem cells from the brain, and providing a renewable population. MSCs grow rapidly in culture, obviating the need for immortalization, and differentiate into neurons using a simple protocol. Moreover, environmentally induced neuronal differentiation in the absence of genetic manipulation may reduce the probability of neoplastic transformation. Finally, the use of adult tissue circumvents the concerns attendant to the use of fetal tissue.
Clonal Analysis To determine whether individual rMSCs exhibited stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, rMSCs were plated at approximately 10 cells/cm2, grown to 50 –150 cells per colony, isolated with cloning cylinders, and transferred to separate wells and finally to individual flasks. Single cells replicated as typical rMSCs and differentiated into NSE-positive neurons after BME treatment. Analysis indicated that each individual clone generated refractile, process-bearing, NSE-positive cells following BME treatment. Undifferentiated rMSCs and transitional cells were evident in each clonal line. Therefore, clones derived from single cells gave rise to both rMSCs and neurons, indicating stem cell characteristics (25). Neurons Differentiate from Human Stromal Cells
ACKNOWLEDGMENTS
To determine whether the neuronal potential of MSCs is unique to rodents, or whether human MSCs (hMSCs) share this ability, hMSCs were isolated from a healthy adult donor and grown in vitro (26). Cells from passage 2 were subjected to the neuronal differentiation protocol and stained for NSE or NF-M expression. After BME treatment, hMSCs exhibited neuronal characteristics and increased NSE expression in a time frame
This work was supported by National Institutes of Health Grant HD23315 and Christopher Reeve Paralysis Foundation Grant IBC-9501. This paper is based on a presentation made at the Focused Workshop on Stem Cell Plasticity sponsored by The Leukemia & Lymphoma Society and the Great Basin Foundation for Biological Research in Santa Barbara, California, on May 4 and 5, 2001. 634
Black and Woodbury
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
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Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. USA 92, 11879 –11883. Johansson, C. B., Momma, S., Clarke, D. L., Risling, M., Lendahl, U., and Frisen, J. (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25–34. Renfranz, P. J., Cunningham, M. G., and McKay, R. D. G. (1991) Region-specific differentiation of hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 6, 713– 729. Gage, F. H., Ray, J., and Fisher, L. J. (1995) Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18, 159 –192. Lundberg, C., Fiueld, P. M., Ajayi, Y. O., Raisman, G., and Bjorklund, A. (1996) Conditionally immortalized neural progenitor cell lines integrate and differentiate after grafting to the adult rat striatum. A combined autoradiographic and electron microscopic study. Brain Res. 737, 295–300. Lundberg, C., Martinez-Serrano, A., Cattaneo, E., McKay, R. D., and Bjorklund, A. (1997) Survival, integration, and differentiation of neural stem cell lines after transplantation to the adult striatum. Exp. Neurol. 145, 342–360. Svendsen, C. N., Caldwell, M. A., Shen, J., ter Borg, M. G., Rosser, A. E., Tyers, P., Karmiuol, S., and Dunnett, S. B. (1997) Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp. Neurol. 148, 135–146. Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U., and Snyder, E. Y. (1998) Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat. Biotechnol. 16, 1033–1039. Bjornson, C. R., Rietze, R. L., Reynolds, B. A., Magli, M. C., and Vescovi, A. L. (1999) Turning brain into blood: A hematopoietic fate adopted by adult neural stem cells in vivo. Science 283, 534 –537. Kopen, G. C., Prockop, D. J., and Phinney, D. G., (1999) Marrow stromal cells migrate throughout the forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. Acad. Sci. USA 96, 10711–10716. Woodbury, D., Schwarz, E. J., Prockop, D. J., and Black, I. B. (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364 –370. Azizi, S. A., Stokes, D., Augelli, B. J., DiGirolamo, C., and Prockop, D. J. (1998) Engraftment and migration of human bone marrow stromal cells implanted in
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
the brains of albino rats-similarities to astrocyte grafts. Proc. Natl. Acad. Sci. USA 95, 3908 –3913. 27. Bruder, S. P., Jaiswal, N., Ricalton, N. S., Mosca, J. D., Kraus, K. H., and Kadiyala, S. (1998) Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin. Orthop. Relat. Res. 355, S247– S256. 28. van Obberghen, E., Kamholz, J., Bishop, J. G., III, Zomzely-Neurath, C., and Lazzarini, R. A. (1988) Human ␥ enolase: Isolation of a cDNA clone and expression in normal and tumor tissues of human origin. J. Neurosci. Res. 19, 450 – 456. 29. Reid, M. M., Wallis, J. P., McGuckin, A. G., Pearson, A. D. J., and Malcolm, A. J. J. (1991) Routine histological compared with immunohistological examination of bone marrow trephine biopsy specimens in disseminated neuroblastoma. Clin. Pathol. 44, 483– 486. 30. Pechumer, H., Bender-Gotze, C., and Ziegler-Heitbrock, H. W. (1993) Detection of neuron-specific eno-
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lase messenger ribonucleic acid in normal human leukocytes by polymerase chain reaction amplification with nested primers. Lab. Invest. 89, 743–749. 31. Carden, M. J., Trojanowski, J. Q., Schlaepfter, W. W., and Lee, V. M. (1987) Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. Neuroscience 7, 3489 –3504. 32. Kosik, K. S., and Finch, E. A. (1987) MAP2 and tau segregate into dendritic and axonal domains after the elaboration of morphologically distinct neurites: An immunocytochemical study of cultured rat cerebrum. J. Neurosci. 7, 3142–3153. 33. Sarnat, H. B., Nochlin, D., and Born, D. E. (1998) Neuronal nuclear antigen (NeuN): A marker of neuronal maturation in early human fetal nervous system. Brain Res. 20, 88 –94. 34. Lendahl, U., Zimmerman, L. B., and McKay, R. D. (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60, 585–595.
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Black and Woodbury
Adult Rat and Human Bone Marrow Stromal Stem Cells Differentiate into Neurons Ira B. Black1 and Dale Woodbury1 Submitted 05/02/01 (Communicated by M. Lichtman, M.D., 05/05/01)
SUMMARY OVERVIEW
INTRODUCTION
Characterization of Stromal Cells Stem cells have been detected in many adult tissues, participating in normal replacement and repair, while undergoing self-renewal (1–11). A subset of bone marrow stem cells constitutes one subtype, capable of differentiating into bone, cartilage, muscle, tendon, fat, and other mesenchymal lineages in vitro (2, 4 –9). They have been termed marrow stromal cells (MSCs). The recent detection of stem cell populations in the central nervous system (CNS) has attracted great attention, since the brain has long been regarded as incapable of regrowth (12, 13). Neural stem cells (NSCs) undergo expansion and differentiate into neurons, astrocytes, and oligodendrocytes in vitro (12, 14 –16). NSCs transplanted into the adult rodent brain survive and differentiate into neurons and glia, raising the possibility of new therapeutic approaches (17–22). However, the inaccessibility of NSCs deep in the brain severely limits clinical utility. A recent report demonstrating that NSCs generate hematopoietic cells in vivo suggests that stem cell populations may be less restricted than was previously believed (23). Evidence that MSCs introduced into the lateral ventricles of neonatal mice can differentiate into astrocytes and neurofilament-containing cells lends support to this contention (24). The present precis briefly describes the differentiation of rat and human MSCs into neurons, and the potential advantages of this approach in the treatment of neurologic disease (25).
Rat mesenchymal stromal cells (rMSCs) were isolated from the femurs of adult rats and propagated in vitro (26). Fluorescent cell sorting at passage 1 demonstrated that the cells were negative for CD11b and CD45, surface markers associated with lymphohematopoietic cells. Consequently, there was no evidence of hematopoietic precursors in the cultures. In contrast, the rMSCs did express CD90, consistent with their undifferentiated state. Initially, with neuronal differentiation, untreated rMSCs were further characterized by staining positively for CD44 and CD71, consistent with previous reports (9, 27). Differentiation into Neurons To induce the neuronal phenotype, rMSCs were maintained in subconfluent cultures in serum-containing medium supplemented with 1 mM -mercaptoethanol (BME) for 24 h (25). To induce neuronal differentiation, the cells were transferred to serum-free medium containing 1–10 mM BME (SFM/BME). Within 60 min of exposure to SFM/BME, changes in morphology of some of the rMSCs were apparent. Responsive cells progressively assumed neuronal morphological traits over the first 3 h. Initially, cytoplasm in the flat rMSCs retracted toward the nucleus, forming a contracted multipolar, cell body, leaving membranous, process-like extensions peripherally
Correspondence and reprint requests to: Ira B. Black. Fax: (732)235-5885. E-mail: [email protected]. 1 Department of Neuroscience and Cell Biology, UMDNJ–Robert Wood Johnson Medical School, 675 Hoes Lane, CABM 342, Piscataway, New Jersey 08854. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
632
Black and Woodbury
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
(0 –90 min). Cells exhibited increased expression of the neuronal marker NSE within 30 min of treatment. Over the subsequent 2 h, cell bodies became increasingly spherical and refractile, exhibiting a typical neuronal perikaryal appearance. Processes continued to elaborate, displaying primary and secondary branches, growth cone-like terminal expansions and putative filopodial extensions. To characterize neuronal differentiation further, we fixed BME-treated cultures after 5 h and stained them for the neuronal marker NSE (25). Unresponsive, flat rMSCs expressed very low, but detectable, levels of NSE protein, consistent with previous detection of minute amounts of protein and/or message in cells of bone marrow origin (28 –30). Progressive transition of rMSCs to a neuronal phenotype coincided with increased expression of NSE. rMSC-derived neurons displayed distinct neuronal morphologies, ranging from simple bipolar to large, extensively branched multipolar cells. Clusters of differentiated cells exhibited intense NSE positivity, and processes formed extensive networks. Western blot analysis confirmed the expression of low levels of NSE protein in uninduced rMSCs. Induction of the neuronal phenotype dramatically increased NSE expression, consistent with the immunocytochemical data. To characterize neuronal identity further, MSCs treated with DMSO/BHA (butylated hydroxyanisole, an antioxidant) were stained for neurofilament-M (NF-M), a neuron-specific intermediate filament that helps initiate neurite elongation (31). We previously found that BME treatment of MSCs elicited increased expression of NF-M in cells exhibiting neuronal morphologies. Most cells displaying rounded cell bodies with processes after DMSO/BHA exposure expressed high levels of NF-M, whereas flat, undifferentiated cells did not. Preadsorption of NF-M antibody with purified NF-M protein abolished staining, establishing specificity (25). We examined DMSO/BHA-treated cultures for the presence of tau, a neuron-specific microtubule-associated protein expressed by differentiating neurons (32). Cells exhibiting a neuronal morphology expressed tau protein in the cell body
as well as in the processes, whereas undifferentiated flat cells were tau-negative (25). To investigate neuronal characteristics further, we examined differentiated cultures for NeuN, a neuron-specific marker expressed in postmitotic cells (33). A subset of cells exhibiting rounded cell bodies and processes stained for NeuN expression, whereas neighboring cells exhibiting distinct neuronal morphologies were NeuN-negative. This observation suggests that a subset of NSE-positive cells are postmitotic neurons. Quantitation of Neuronal Differentiation With this induction protocol, a variable number of cells underwent neuronal differentiation, although the response generally exceeded 50%. To optimize differentiation, we modified the preinduction protocol. Addition of bFGF (10 ng/ml) and elimination of BME from the preinduction medium increased the proportion of cells exhibiting neuronal traits, and the response was more consistent within and between experiments. To quantitate this response, rMSCs were treated with the modified preinduction protocol, and induced to differentiate with DMSO/BHA. Cells were fixed after 5 h, stained for the neuronal markers NSE and NF-M, and the percentage of neuronal cells was determined. The majority of rMSCs treated in this manner exhibited neuronal morphologies and stained positively for NSE (78.2 ⫾ 2.3%) and NF-M (79.2% ⫾ 2.5%) expression (25). Long-Term Differentiation A dramatic aspect of rMSC neuronal differentiation is the rapidity of the response. As a result, our analysis focused on changes that occurred within the first 5 h of differentiation. However, long-term differentiation of these cells will be important to our understanding of this process. To address this issue, we monitored expression of the nestin gene product in rMSC-derived neurons at 5 h, 1 day, and 6 days postdifferentiation. Nestin, an intermediate filament protein, is expressed in neuroepithelial neuronal precursor 633
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
stem cells, and its expression decreases with neuronal maturation (34). A subset of rMSC-derived neurons expressed high levels of nestin protein at 5 h, and the proportion of nestin-positive cells decreased with time. By 6 days postinduction, there was no detectable nestin expression in any rMSC-derived neurons, consistent with ongoing maturation with time (25). We compared the time course of expression of the transitional trait, nestin, to that of a mature phenotypic character, trkA, the biologically active nerve growth factor receptor. TrkA was detectable at 5 h, the earliest time examined and, in contrast to nestin, persisted unchanged through 6 days (25).
Black and Woodbury
similar to that observed for rMSCs. Contracted cell bodies elaborated processes and stained intensely for NSE expression within 3 h. Transitional cells were also evident. Many processes elaborated by hMSC-derived neurons exhibited terminal bulbs, which may represent growth cones. These cells also expressed NF-M, consistent with neuronal differentiation. PERSPECTIVE Our observations suggest that intrinsic genomic mechanisms of commitment, lineage restriction, and cell fate are plastic. The environment apparently can evoke the expression of pluripotentiality that far exceeds traditional fate restrictions of cells derived from the classical embryonic germ layers. The adult cells are both self-renewing and pluripotential (2–9), thereby satisfying many of the criteria of a stem cell population. The use of MSCs to generate neurons for transplantation confers a number of potential therapeutic advantages. Autologous transplantation eliminates the hazards of immunorejection, and obviates the need for toxic immunosuppressive agents. The bone marrow is a safe, accessible source, overcoming the risks of obtaining stem cells from the brain, and providing a renewable population. MSCs grow rapidly in culture, obviating the need for immortalization, and differentiate into neurons using a simple protocol. Moreover, environmentally induced neuronal differentiation in the absence of genetic manipulation may reduce the probability of neoplastic transformation. Finally, the use of adult tissue circumvents the concerns attendant to the use of fetal tissue.
Clonal Analysis To determine whether individual rMSCs exhibited stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, rMSCs were plated at approximately 10 cells/cm2, grown to 50 –150 cells per colony, isolated with cloning cylinders, and transferred to separate wells and finally to individual flasks. Single cells replicated as typical rMSCs and differentiated into NSE-positive neurons after BME treatment. Analysis indicated that each individual clone generated refractile, process-bearing, NSE-positive cells following BME treatment. Undifferentiated rMSCs and transitional cells were evident in each clonal line. Therefore, clones derived from single cells gave rise to both rMSCs and neurons, indicating stem cell characteristics (25). Neurons Differentiate from Human Stromal Cells
ACKNOWLEDGMENTS
To determine whether the neuronal potential of MSCs is unique to rodents, or whether human MSCs (hMSCs) share this ability, hMSCs were isolated from a healthy adult donor and grown in vitro (26). Cells from passage 2 were subjected to the neuronal differentiation protocol and stained for NSE or NF-M expression. After BME treatment, hMSCs exhibited neuronal characteristics and increased NSE expression in a time frame
This work was supported by National Institutes of Health Grant HD23315 and Christopher Reeve Paralysis Foundation Grant IBC-9501. This paper is based on a presentation made at the Focused Workshop on Stem Cell Plasticity sponsored by The Leukemia & Lymphoma Society and the Great Basin Foundation for Biological Research in Santa Barbara, California, on May 4 and 5, 2001. 634
Black and Woodbury
Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 632– 636 doi:10.1006/bcmd.2001.0423, available online at http://www.idealibrary.com on
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Hay, E. (1966) Regeneration. Holt, Rinehart and Winston, New York. Kuznetsov, S. A., Friedenstein, A. J., and Robey, P. G. (1997) Factors required for bone marrow stromal fibroblast colony formation in vitro. Br. J. Haematol. 97, 561–570. Owens, M. E., and Friedenstein, A. J. (1988) Cell and molecular biology of vertebrate hard tissues. Ciba Found. Symp. 136, 42– 60. Caplan, A. I. (1991) Mesenchymal stem cells. J. Orthop. Res. 9, 641– 650. Pereira, R. F., Halford, K. W., O’Hara, M. D., Leeper, D. B., Sokolov, B. P., Pollard, M. D., Bagasra, O., and Prockop, D. J. (1995) Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc. Natl. Acad. Sci. USA 92(11), 4857– 4861. Prockop, D. J. (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71– 74. Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G., and Mavilio, F. (1998) Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528 –1530. Majumdar, M. K., Thiede, M. A., Mosca, J. D., Moorman, M., and Gerson, S. L. (1998) Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J. Cell. Physiol. 176, 57– 66. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. McKay, R. D. (1999) Brain stem cells change their identity. Nat. Med. 5, 261–262. Lemischka, I. (1999) Searching for stem cell regulatory molecules. Some general thoughts and possible approaches. Ann. N.Y. Acad. Sci. 872, 274 –288. Reynolds, B. A., and Weiss, S. (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710. Richards, L. J., Kilpatrick, T. J., and Bartlett, P. F. (1992) De novo generation of neuronal cells from the adult mouse brain. Proc. Natl. Acad. Sci. USA 89, 8591– 8595. Vescovi, A. L., Reynolds, B. A., Fraser, D. D., and Weiss, S. (1993) bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11, 951–966. Gage, F. H., Coates, P. W., and Palmer, T. D. (1995)
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