The Study On The Biological Characteristics Of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells, Differentiation Into Neurocyte-like Cells, And Transplantation Into Experimental Traumatic Brain Injury To Improve Neurological Function

Stem cells are characterized by a capacity to self-renew and to generate progeny capable of differentiating into multiple yet distinct other cell lineages. Neural Stem cells (NSCs) derived from embryo has been the hope for treating the disease of central nervous system, including neurodegenerative disorders, traumatic brain injury and stroke, but their utility is severely limited by lack of accessibility and restricted by ethics. Recent observations reveal that some adult stem cells might have greater plasticity than were previously thought, especially, the bone marrow mesenchymal stem cells (bMSCs) has been successfully cultured in vitro and can differentiate into cells that are not part of their normal repertoire: skeletal, muscle, hepatocytes, glia and neuron. However, the use of bMSCs is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferation/ differentiation capacity with age, their therapeutic potential might be diminished as well, which makes it necessary to search for adequate alternative sources of these cells for autologous and allogenic applications."Human Umbilical cord blood (HUCB)" is the blood that remains in placenta and umbilical cord after birth. HUCB is an excellent alternative source of hematopoietic stem/progenitor cells for clinical-scale allogeneic transplantation. Essential preclinical studies proved a higher percentage of CD34~+ CD38~- cells in HUCB compared with BM, suggesting that more primitive progenitor may beabundant in neonatal blood. The same might apply for the presence of MSCs or progenitor cells. However, previous attempt to isolate MSCs form HUCB have either failed or have demonstrated a low frequency of mesenchymal progenitor in HUCB of full-term deliveries. Therefore, the first goal of the present study was to verify whether cells with MSCs traits can be isolated from full-term HUCB units, and made an attempt to establish the method of culture, purification and expansion of HUCB-derived MSCs in vitro. Second, those cells were compared with BM-derives MSCs to assess potential similarities or differences in biological characteristic, including induced these cells to differentiate into neurons and astrocytes. In addition we investigated the possibility of repairing the nervous system by transplanting HUCB-derived MSCs transplants after traumatic brain injury.Part One: Investigation on the biological character and establishment the cells culture system of HUCB-derived mesenchymal stem cells in vitroHUCB has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether MSCs are invariably present in HUCB of full-term deliveries, in this study, we compared two methods for primary culture of HUCB-derived MSCs and have successfully established the method of isolation, purification and expansion of HUCB-derived MSCs in vitro. 1. We used the standard method of culture the MSCs, which was first suggested by Friendenstein nearly 40 years ago, and has been successfully used in culture bMSCs in vitro. HUCB was collected after obtaining written informed consent. All samples were prepared from full term deliveries scheduled for cesarean section by puncturing umbilical cord vein. Mononuclear cells (MNCs) were isolated through standard density gradient (Ficoll-Paque), and then cultured in L-DMEM (Low glucose-DMEM) containing 10% fetal bovine serum, 1% L-glutamine, lOng/ml b-FGF,10ng/ml FGF, 1 % Pen-strep, and seeded at a concentration of lxlO6/ml cells/cm2. Cultures were incubated at 37°C, 5%CO2 and a fully humidified atmosphere. After culture for 48 -72h for the culturemedium was replaced and non-adherent cells were removed. The medium was then changed every 3-4 d. After 14 d, cells in the developed adherent layer were used for the experiments. At 80—90% confluence, cells were harvested with 25% trypsin for 5 min at 37°C and were passaged at a ratio of 1:2. 2. In our culture system, the collection and separation of MNCs-derived from HUCB was identical with above. The mononuclear cell layer was resuspended in the complete culture medium to a concentration of lxlO8 cells/ml. The culture medium consisted of Mesencult?, supplemented with 1% L-Glutamine, lOOunits/ml Pen-strep, and adjusted the medium pH with HEPES, which rendered a final pH of around 7.0. The cells were seeded in 25T cell culture flask which had been coated with FBS and incubated at 37°C, 5%CC>2, in a fully humidified atmosphere for 16 days. Approximately 7 days later, when cells in the developing adherent layer were appeared, non-adherent cells were washed off with medium changes. Half of Culture medium changes were carried out every 3—5 days thereafter. Once adherent cells reached approximately 80—90% confluent, they were detached and re-plated at 1:2 under the same culture conditions.Our results show: using the standard method of culture the MSCs, most of the derived adherent cells morphology was heterogeneous and no obvious CFU were observed. They reached confluence after about 2 weeks, large, round-shaped cells and fibroblast-life cells were prevalent, although these heterogeneous adherent cells could reach confluence in primary passage, their proliferation capacity was limited, they were never passaged beyond the third passage;Through our exploration, HUCB-derived mononuclear cells of full-term deliveries when cultured in the suitable medium and higher density seeding with coating culture flask, were able to generate adherent cells, which exhibited either an osteoclast- or mesenchymal-like phenotype. With the conditional medium and subculture, the adherent cells can be purified, and gave rise to a well-established layer of MSCs. They sustain prolonged self-renewal, maintaining homogeneous characteristics for over 20 passages.We examined the proliferation characteristics of HUCB-derived MSCs, and cell growth curves were depicted through cell count and contrast at the primary culture and the fifth passage. The confluence of primary cultures was reachedafter approximately two weeks of culture. With the heterogeneous cell decreasing gradually, the subculture cells of MSC grew rapidly, which proliferated with a population-doubling time of 3-4 days and reached a confluent growth-arrested after 7-8 days. These cells expressed CD29, CD44, and CD105, well-known MSC-specific antigens. They were uniformly negative for expression of CD34, CD45, CD106, and HLA-DR, which are consistent with the finding for bMSCs. All data above indicate that the post-purified cells derived from HUCB exhibit the phenotype of MSCs.Part two: Study on differentiation into neurocyte-like cells of HUCB-derivedMSCsThe ability to selectively produce one or more differentiated cell types at will from multipotential stem cells would be of profound clinical importance, as it would enable the specific replacement of damaged/dysfunctional cell types within the body, potentially curing numerous diseases. However, our understanding of the processes that regulate the differentiated direction controlling the formation of functional tissues from MSCs is still in limited. In this study, we will explore the differentiation potential of HUCB-derived MSCs and how to improve the proportion of it differentiation into neuron-like cells in vitro.The differentiation of MSCs was assessed in the post fifth-passage cultures. Cells were seeded at a density of lxlO4 cells/cm2, combinative induced for six days in DMEM/F12 plug 5% FBS, lQumol/L RA, lOng/ml BDNF and lOng/ml CNTF. After four days, cells were fixed for immunofluorescence detection of neuron specific antigens, and the neuron markers analyzed by RT-PCR.We used the neural induction medium including: RA, BDNF and CNTF, after six days of differentiation 70% cells in the dish acquired the morphology of neuron like cells, which exhibited a multipolar, refractile cell body bearing many process-like extensions. The cell body condensed and became increasingly spherical, refractile and acquired a typical neuronal perikaryal appearance. Immunofluorescence staining for neural markers was performed to confirm the neural marker expression in differentiated MSCs. On the sixth day in culture, NSE,NF, and GFAP immunoreacting cells were found. Some of NSE immunoreacting cells elaborating multiple processes showed mature neuronal morphologies. Immunofluorescence assays on undifferentiated cells stained negative for NSE, NF and GFAP (not shown). RT-PCR analysis revealed expression of NSE, neurofilament subunit M (NF-M) (marker gene of neuron), and GFAP (marker of glia) ,while B-actin served as the loading control.Part three: HUCB-derived MSCs transplants promote neurological functional recovery after traumatic brain injuryTraumatic brain injury (TBI) is one of the leading causes of mortality and morbidity in our countries, most people suffer the debilitating socioeconomic costs associated with the physical, cognitive, and psychosocial sequelae of TBI. The nervous system, unlike many other tissues, has a limited capacity for self-repair, mature nerve cells lack the ability to regenerate, and neural stem cells although they exist even in the adult brain, have a limited ability to generate new functional neurons in response to injury. For this reason, there is great interest in the possibility of repairing the nervous system by transplanting cells that can replace those lost through damage or disease. The most promising neural transplant results have been obtained with neurons from human fetuses. However, limited amounts of human fetal tissue are available for grafting purposes. The use of human fetal tissue also raises questions as to the standardization, viability and purity of the cell material, and the use of aborted fetuses for medical purposes is also ethically controversial. Recently, the transplantation or administration of bone mesenchymal stem cells (bMSCs) for replacement therapies of central nervous system disease and injury has been reported. Besides bone marrow, human umbilical cord blood has been recognized as another source of stem cells.In this study, we test the hypothesis that HUCB-derived MSCs delivered to brain after TBI via located injection to express neural markers and promote neurological functional improvement. The HUCB-derived MSCs were labeled by bis-benzimide, a nuclear fluorescence dye, over 24 h, and were stereotactically transplanted into the brain of the mice. All animals were divided into four groups:...