脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

Comparative analysis of human UCB and adipose tissue derived mesenchymal stem cells for their differentiation potential into brown and white adipocytes

The differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into brown and white adipocytes in comparison to Adipose tissue derived MSCs (AD-MSCs) were investigated in order to characterize their potency for future cell therapies. MSCs were isolated from ten UCB samples and six liposuction materials. MSCs were differentiated into white and brown adipocytes after characterization by flow cytometry. Differentiated adipocytes were stained with Oil Red O and hematoxylin/eosin. The UCP1 protein levels in brown adipocytes were investigated by immunofluoresence and western blot analysis. Cells that expressed mesenchymal stem cells markers (CD34?, CD45?, CD90+ and CD105+) were successfully isolated from UCB and adipose tissue. Oil Red O staining demonstrated that white and brown adipocytes obtained from AD-MSCs showed 85 and 61% of red pixels, while it was 3 and 1.9%, respectively for white and brown adipocytes obtained from UCB-MSCs. Fluorescence microscopy analysis showed strong uncoupling protein 1 (UCP1) signaling in brown adipocytes, especially which were obtained from AD-MSCs. Quantification of UCP1 protein amount showed 4- and 10.64-fold increase in UCP1 contents of brown adipocytes derived from UCB-MSCs and AD-MSCs, respectively in comparison to undifferentiated MSCs (P?<?0.004). UCB-MSCs showed only a little differentiation tendency into adipocytes means it is not an appropriate stem cell type to be differentiated into these cell types. In contrast, high differentiation efficiency of AD-MSCs into brown and white adipocytes make it appropriate stem cell type to use in future regenerative medicine of soft tissue disorders or fighting with obesity and its related disorders.     

Regenerative medicine for Parkinson’s disease using differentiated nerve cells derived from human buccal fat pad stem cells

The purpose of this study was to evaluate the utility of human adipose stem cells derived from the buccal fat pad (hBFP-ASCs) for nerve regeneration. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons. PD is a candidate disease for cell replacement therapy because it has no fundamental therapeutic methods. We examined the properties of neural-related cells induced from hBFP-ASCs as a cell source for PD treatment. hBFP-ASCs were cultured in neurogenic differentiation medium for about 2 weeks. After the morphology of hBFP-ASCs changed to neural-like cells, the medium was replaced with neural maintenance medium. Cells differentiated from hBFP-ASCs showed neuron-like structures and expressed neuron markers (β3-tubulin, neurofilament 200, and microtubule-associated protein 2), an astrocyte marker (glial fibrillary acidic protein), or dopaminergic neuron-related marker (tyrosine hydroxylase). Induced neural cells were transplanted into a 6-hydroxydopamine (6-OHDA)-lesioned rat hemi-parkinsonian model. At 4 weeks after transplantation, 6-OHDA-lesioned rats were subjected to apomorphine-induced rotation analysis. The transplanted cells survived in the brain of rats as dopaminergic neural cells. No tumor formation was found after cell transplantation. We demonstrated differentiation of hBFP-ASCs into neural cells, and that transplantation of these neural cells improved the symptoms of model rats. Our results suggest that neurons differentiated from hBFP-ASCs would be applicable to cell replacement therapy of PD.     

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs’ protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.     

Immunophenotypic characterization and tenogenic differentiation of mesenchymal stromal cells isolated from equine umbilical cord blood

Mesenchymal stem cells (MSCs) isolated from umbilical cord blood (UCB) in equines have not been well characterized with respect to the expression of pluripotency and mesenchymal markers and for tenogenic differentiation potential in vitro. The plastic adherent fibroblast-like cells isolated from 13 out of 20 UCB samples could proliferate till passage 20. The cells expressed pluripotency markers (OCT4, NANOG, and SOX2) and MSC surface markers (CD90, CD73, and CD105) by RT-PCR, but did not express CD34, CD45, and CD14. On immunocytochemistry, the isolated cells showed expression of CD90 and CD73 proteins, but tested negative for CD34 and CD45. In flow cytometry, CD29, CD44, CD73, and CD90 were expressed by 96.36??±?1.28%, 93.40??±?0.70%, 73.23??±?1.29% and 46.75??±?3.95% cells, respectively. The UCB-MSCs could be differentiated to tenocytes by culturing in growth medium supplemented with 50 ng/ml of BMP-12 by day 10. The differentiated cells showed the expression of mohawk homeobox (Mkx), collagen type I alpha 1 (Col1α1), scleraxis (Scx), tenomodulin (Tnmd) and decorin (Dcn) by RT-PCR. In addition, flow cytometry detected tenomodulin and decorin protein in 95.65?±?2.15% and 96.30?±?1.00% of differentiated cells in comparison to 11.30?±?0.10% and 19.45?±?0.55% cells, respectively in undifferentiated control cells. The findings support the observation that these cells may be suitable for therapeutic applications, including ruptured tendons in racehorses.     

Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells

Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have multi-lineage differentiation potential, thus highlighting the feasibility of using UCB-MSCs as a valuable source of stem-cells for cell-based therapy. However, there are no well-defined markers for assessment of the multi-potency of UCB-MSCs. Thus, we focused on the identification of suitable markers by examining cell surface protein expressions of UCB-MSCs as their multi-lineage differentiations progressed. The expression of CD105, one of the cell surface proteins, was significantly decreased in differentiated osteoblasts, chondrocytes, adipocytes, and respiratory epithelium, and the portion of CD105-positive cells from 99.4 ± 0.1% to 3.5 ± 1.4%, 3.5 ± 2.3%, 16.7 ± 3.6%, and 2.1 ± 1.5%, respectively. As to such indicators as alkaline phosphatase (ALP), glycosaminoglycan (GAG), oil Red O, and surfactant protein C (SPC), they showed increases, confirming differentiation of UCB-MSCs into osteoblasts, chondrocytes, adipocytes, and respiratory epithelium. This is the first study to demonstrate a negative correlation between expression of CD105 over the time course of multi-lineage differentiation and the degree of differentiation of UCB-MSCs. We propose that CD105 is a useful novel marker to characterize differentiation status of isolated human UCB-MSCs, which will be useful to facilitate the application of such cells in stem-cell therapy.     

Immunocytochemical analysis of valproic acid induced histone H3 and H4 acetylation during differentiation of rat adipose derived stem cells into neuron-like cells

Valproic acid (VPA) is an inhibitor of histone deacetylases (HDACs) that can regulate differentiation and proliferation of stem cells by epigenetic mechanisms. We investigated VPA induced histone H3 and H4 acetylation in adipose derived stem cells (ADSCs) transdifferentiated into neuron-like cells (NLCs). Rat ADSCs were transdifferentiated into neural stem cells (NSCs) that had been generated from neurospheres. The NSCs were differentiated into NLCs by induction with different concentrations of VPA at 24, 48 and 72 h. The NLCs were evaluated using anti-H3 and -H4 antibodies, and ADSCs, NSCs and NLCs were evaluated using immunofluorescence. The ADSCs were immunoreactive to CD90 and CD49d, but not to CD45 and CD31. Both the neurospheres and NSCs were immunostained with nestin and neurofilament 68. The neurospheres expressed Musashi1, Sox2 and Neu N genes as determined by RT-PCR. Our dose-response study indicated that the optimal concentration of VPA was 1 mM at 72 h. Histone acetylation levels of H3 and H4 immunostaining intensities in NLCs were significantly greater than for ADSCs and NSCs. VPA alters H4 and H3 acetylation immunoreactivities of ADSCs transdifferentiated into NLCs.     

Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague–Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.     

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Background aimsAmniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro.MethodsAF was harvested during routine pre-natal amniocentesis at 14–16 weeks of pregnancy. AF sample pellets were plated in α-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction.ResultsTwenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na⁺ channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na⁺ channels.ConclusionsThese data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.     

Clonal In Vitro Analysis of Neurotrophin Receptor p75-Immunofluorescent Cells Reveals Phenotypic Plasticity of Primary Rat Olfactory Ensheathing Cells

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75^NTR)-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell?) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75^NTR expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75^NTR at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.     

A Comparative Study to Evaluate Myogenic Differentiation Potential of Human Chorion versus Umbilical Cord Blood-derived Mesenchymal Stem Cells

Objective

Musculodegenerative diseases threaten the life of many patients in the world. Since drug administration is not efficient in regeneration of damaged tissues, stem cell therapy is considered as a good strategy to restore the lost cells. Since the efficiency of myogenic differentiation potential of human Chorion- derived Mesenchymal Stem Cells (C-MSCs) has not been addressed so far; we set out to evaluate myogenic differentiation property of these cells in comparison with Umbilical Cord Blood- derived Mesenchymal Stem Cells (UCB-MSCs) in the presence of 5-azacytidine.

诱导人脐带间充质干细胞分化为视网膜色素上皮样细胞的研究

目的：探讨用猪视网膜色素上皮细胞（RPE）条件培养基诱导hUCMSCs分化为RPE样细胞的方法。方法通过流式细胞技术鉴定hUCMSCs的表面标记分子;通过诱导hUCMSCs分化成为脂肪、骨和软骨细胞确定其多系分化能力;将hUCMSCs培养在猪RPE的条件培养液中并添加诱导因子来诱导hUCMSCs向RPE细胞分化。对照组与处理组Q-PCR结果采用t检验比较。结果 hUCMSCs表达CD105、CD90、CD73、CD44和CD29,但不表达CD34,CD45和MHCII等分子标记,在成脂、成骨、成软骨分化培养基中可分化为脂肪、骨和软骨细胞。单独使用猪RPE条件培养液不能有效诱导hUCMSCs向RPE细胞分化,但联合应用猪RPE条件培养液和诱导因子（视黄酸,activin-A和人重组骨形成蛋白-7）可有效诱导hUCMSCs分化为RPE样细胞,RPE细胞标记分子RPE65、Mitf和Ck8/18的基因表达量分别提高了2.1±0.4、6.8±1.3和2.5±0.3倍（P〈0.05）。诱导产生的RPE样细胞呈多边形,但不含色素颗粒。结论猪RPE条件培养液联合诱导因子可有效诱导hUCMSCs分化为RPE样细胞,可能会为治疗视网膜变性疾病提供合适的种子细胞。     

Isolation,characterisation and comparative analysis of human umbilical cord vein perivascular cells and cord blood mesenchymal stem cells

Perivascular cells are known to be ancestors of mesenchymal stem cells (MSCs) and can be obtained from heart, skin, bone marrow, eye, placenta and umbilical cord (UC). However detailed characterization of perivascular cells around the human UC vein and comparative analysis of them with MSCs haven’t been done yet. In this study, our aim is to isolate perivascular cells from human UC vein and characterize them versus UC blood MSCs (UCB-MSCs). For this purpose, perivascular cells around the UC vein were isolated enzymatically and then purified with magnetic activated cell sorting (MACS) method using CD146 Microbead Kit respectively. MSCs were isolated from UCB by Ficoll density gradient solution. Perivascular cells and UCB-MSCs were characterized by osteogenic and adipogenic differentiation procedures, flow cytometric analysis [CD146, CD105, CD31, CD34, CD45 and alpha-smooth muscle actin (α-SMA)], and immunofluorescent staining (MAP1B and Tenascin C). Alizarin red and Oil red O staining results showed that perivascular cells and MSCs had osteogenic and adipogenic differentiation capacity. However, osteogenic differentiation capacity of perivascular cells were found to be less than UCB-MSCs. According to flow cytometric analysis, CD146 expression of perivascular cells were appeared to be 4.8-fold higher than UCB-MSCs. Expression of α-SMA, MAP1B and Tenascin-C from perivascular cells was determined by flow cytometry analysis and immunfluorescent staining. The results appear to support the fact that perivascular cells are the ancestors of MSCs in vascular area. They may be used as alternative cells to MSCs in the field of vascular tissue engineering.     

Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro

Results of recent investigations have demonstrated the plasticity of mesenchymal stem cells (MSC) can differentiate into neural lineages. In this study, we explored the experimental condition of differentiation into neuron-like cells or rhodopsin (RHOS)-positive cells induced by epidermal growth factor (EGF) and taurine in vitro and to investigate their biological characteristics. MSC were obtained from umbilical cord blood (UCB) of term deliveries. Cultured cells were treated with Dulbecco’s modified Eagle’s medium/F12 (pH 7.0–7.2) supplemented with 30 ng/ml EGF. After the third cell passage, the cells were trysinized and analyzed with a flow cytometer using the following monocloned antibodies: CD90, CD29, CD34, CD44, and CD45. Taking another MSC of the third passage, its basal medium was replaced with alpha minimum essential medium supplemented with taurine (50 μmol/L). Cells were cultured for an additional 8–10 d, fixed, and then immunocytochemically analyzed. Primary antibodies included the following: neuron-specific enolase (NSE), RHOS, and nestin. In our study, we isolated a cell population derived from UCB, which possesses morphological characteristics similar to those of MSC isolated from bone marrow. In the cytometric analysis, MSC did not present labeling for the hematopoietic line (CD34 and CD45) and were positive for CD29, CD44, and CD90. After induction by taurine, 80.5 ± 16.2% of the cell population expressed NSE, 36.8 ± 9.6% expressed RHOS, and 29.6 ± 9.3% expressed Nestin, while only 7.9 ± 3.5% expressed NSE in the control group. This study demonstrates that partial MSC induced by taurine and EGF can differentiate into neuron-like cells or RHOS-positive cells in vitro, which may provide a promising therapeutic strategy for the treatment of some forms of retinal degeneration.     

Functional Analysis of Neuron-like Cells Differentiated from Neural Stem Cells Derived from Bone Marrow Stroma Cells in vitro

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in “NSCs medium”. Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (γ) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na⁺ and K⁺ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.     

Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds

Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1 μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15 days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.     

Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC–MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm²) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO₂. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications.     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.