ABSTRACT
<p><b>OBJECTIVE</b>To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.</p><p><b>METHODS</b>The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.</p><p><b>RESULTS</b>A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.</p><p><b>CONCLUSION</b>The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.</p>
Subject(s)
Female , Humans , Infant, Newborn , Male , Pregnancy , Antigens, CD , Allergy and Immunology , Carrier Proteins , Genetics , Cell Differentiation , Physiology , Cells, Cultured , Cytokines , Genetics , Flow Cytometry , Glial Fibrillary Acidic Protein , Metabolism , Immunohistochemistry , Intermediate Filament Proteins , Genetics , Mesenchymal Stem Cells , Allergy and Immunology , Metabolism , Physiology , Nerve Tissue Proteins , Genetics , Nestin , Neurofilament Proteins , Metabolism , Neurons , Metabolism , Physiology , Reverse Transcriptase Polymerase Chain Reaction , Tubulin , Metabolism , Umbilical Cord , Cell BiologyABSTRACT
<p><b>BACKGROUND</b>The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells.</p><p><b>METHODS</b>MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.</p><p><b>CONCLUSIONS</b>MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.</p>