• Bicknese AR, Goodwin HS, Quinn CO, Henderson VC, Chien SN and Wall DA (2002). Human umbilical cord blood cells can be induced to express markers for neurons and glia. Cell Transplant 11:261-4. Summary: Rare cells are present in human umbilical cord blood that do not express the hematopoietic marker CD45 and in culture do not produce cells of hematopoietic lineage. These umbilical cord multipotent stem cells (UC-MC) behave as multilineage progenitor cells (stem cells) and can be expanded in tissue culture. Exposure to basic fibroblast growth factor (bFGF) and human epidermal growth factor (hEGF) for a minimum of 7 days in culture induces expression of neural and glial markers. Western immunoblots demonstrate expression of both beta-tubulin III and glial fibrillary acidic protein (GFAP). Immunocytochemistry of the cells showed intense labeling to both compounds on the intracellular cytoskeleton. The oligodendrocyte cell surface marker galactocerebroside (Gal-C) was present on most cells. Many cells show dual labeling, expressing both neuronal and glial markers. Department of Neurology, Saint Louis University, and Cardinal Glennon Children's Hospital, MO 63110, USA. bicknese@slu.edu
• Buzanska L, Machaj EK, Zablocka B, Pojda Z and Domanska-Janik K (2002). Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci 115:2131-8. Summary: Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells. Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego St. 02-106 Warsaw, Poland.
• Goodwin HS, Bicknese AR, Chien SN, Bogucki BD, Quinn CO and Wall DA (2001). Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7:581-8. Summary: The stromal cell population in bone marrow has been the focus of much attention since it has been shown that this cell population can be expanded and differentiated into cells with the phenotype of bone, cartilage, muscle, stroma, neural, and fat cells. We evaluated umbilical cord blood (UCB) for the presence of these cells. From the mononuclear fraction of UCB, we demonstrated the presence of a subset of cells that have been maintained in continuous culture for more than 6 months (>10 passages). These adherent cell populations express adhesion molecules CD13+, CD29+, and CD44+, but not antigens of hematopoietic differentiation. Exposure of these cells to osteogenic agents resulted in an increase in expression of alkaline phosphatase and the appearance of hydroxyapatite nodules by Von Kossa staining. Incubation with adipogenic agents resulted in morphological change and staining with Oil Red O. In addition, when exposed to basic fibroblast growth factor and human epidermal growth factor the cells underwent changes consistent with cells of neural origin. These changes were demonstrated by a combination of immunofluorescent labeling and Western immunoblots for neural-specific markers. Thus, similar to what has been previously reported with bone marrow, cord blood contains a population of cells that can be expanded in culture and are able to express the phenotype of multiple lineages. Cord blood multilineage cells are slower to establish in culture, have a lower precursor frequency and a lower level of bone antigen expression, and lack constitutive expression of neural antigens when compared to bone marrow, suggesting a more primitive population. Cord blood may prove to be a new source of cells for cellular therapeutics for stromal, bone, and, potentially, neural repair. Departments of Pediatrics, Pediatric Research Institute, Cardinal Glennon Children's Hospital, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
• Ha Y, Choi JU, Yoon DH, Yeon DS, Lee JJ, Kim HO and Cho YE (2001). Neural phenotype expression of cultured human cord blood cells in vitro. Neuroreport 12:3523-7. Summary: Neural stem cells have been proposed as useful vectors for treating diseases in the CNS, but their utility is severely limited by lack of accessibility. Brain development is ongoing extensively in early postnatal life. However, it is unclear whether stem cells that differentiate into neurons exist in the blood during early postnatal life. We showed in this experiment that neural markers (NeuN, neurofilament, MAP2, GFAP) are expressed and long cytoplasmic processes are elaborated in the cultured human cord blood monocytes prepared from newborn umbilical blood. These results suggest that stem cells in human cord blood may be potential sources of neurons in early postnatal life. We suggest that the neonatal blood system functions as a circulating pool of different types of stem cell. Department of Neurosurgery, Seoul District Hospital, 165 Sokyuk-dong, Jongro-gu, Seoul 110-200, Korea.
• Hou L, Cao H, Wei G, Bai C, Zhang Y, Wu Z and Pei Xt X (2002). Study of in vitro expansion and differentiation into neuron-like cells of human umbilical cord blood mesenchymal stem cells. Zhonghua Xue Ye Xue Za Zhi 23:415-9. Summary: OBJECTIVE: To explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro. METHODS: Human cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods. RESULTS: The number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a numbe of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry. CONCLUSION: MSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells. Chinese Academy of Military Medical Science, Beijing 100850, China.
• Sanchez-Ramos JR (2002). Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 69:880-93. Summary: Under experimental conditions, tissue-specific stem cells have been shown to give rise to cell lineages not normally found in the organ or tissue of residence. Neural stem cells from fetal brain have been shown to give rise to blood cell lines and conversely, bone marrow stromal cells have been reported to generate skeletal and cardiac muscle, oval hepatocytes, as well as glia and neuron-like cells. This article reviews studies in which cells from postnatal bone marrow or umbilical cord blood were induced to proliferate and differentiate into glia and neurons, cellular lineages that are not their normal destiny. The review encompasses in vitro and in vivo studies with focus on experimental variables, such as the source and characterization of cells, cell-tracking methods, and markers of neural differentiation. The existence of stem/progenitor cells with previously unappreciated proliferation and differentiation potential in postnatal bone marrow and in umbilical cord blood opens up the possibility of using stem cells found in these tissues to treat degenerative, post-traumatic and hereditary diseases of the central nervous system. Center of Aging and Brain Repair, University of South Florida and James Haley VA Hospital Health Science Center, Tampa, Florida 33612, USA. jsramos@hsd.usf.edu
• Sanchez-Ramos JR, Song S, Kamath SG, Zigova T, Willing A, Cardozo-Pelaez F, Stedeford T, Chopp M and Sanberg PR (2001). Expression of neural markers in human umbilical cord blood. Exp Neurol 171:109-15. Summary: A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders. Center for Aging and Brain Repair, Department of Neurology, University of South Florida College of Medicine, Tampa, Florida, USA.
• Song S and Sanchez-Ramos J (2002). Preparation of neural progenitors from bone marrow and umbilical cord blood. Methods Mol Biol 198:79-88. Summary: Department of Neurology, Center for Aging and Brain Repair, College of Medicine, University of South Florida, Tampa, FL, USA.
• Zigova T, Song S, Willing AE, Hudson JE, Newman MB, Saporta S, Sanchez-Ramos J and Sanberg PR (2002). Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 11:265-74. Summary: Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III beta-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain. Center for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa 33612, USA. tzigova@hsc.usf.edu