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.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Quantity and proliferation rate of mesenchymal stem cells in human cord blood during gestation

UCB (umbilical cord blood) as a resource of MSCs (mesenchymal stem cells) is widely accepted, but the quantity and characteristics of UCB-MSCs from different gestational ages have not been well studied. We have quantified the number of MSCs in UCB at different gestational ages using a multi-colour flowcytometer and compared the cell proliferation rates of these UCB-MSCs. Defining MSCs as CD44+/CD105+/CD34-/CD45 population, their numbers declined in the UCB at the gestational age. Proliferation rates were significantly higher in UCB before term than at full term. Non-full term UCB samples may be a better source of MSCs.     

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.     

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。     

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.     

A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34(+) cells isolated from umbilical cord blood

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ～15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.     

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.     

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l -lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.     

Isolation,expansion and characterisation of mesenchymal stem cells from human bone marrow,adipose tissue,umbilical cord blood and matrix: a comparative study

The multipotent and immunosuppressive capacities of mesenchymal stem cells (MSCs) attract several scientists worldwide towards translational research focusing on treatment of diseases including liver failure. Though MSC’s have been isolated from different sources, researchers do not concur on the best source for expansion and clinical translation. In this study, we have compared the isolation, proliferation and expansion of MSCs from umbilical cord blood (UCB), Wharton’s Jelly (WJ), bone marrow (BM) and adipose tissue (AT). MSCs were isolated by density gradient separation from UCB, BM and AT and by both enzymatic and explant method for WJ. The MSCs are characterized by their ability to adhere to plastic, expression of positive (CD105, CD73, CD90, CD29, CD44) and negative (CD45, CD14, CD34) markers by flow cytometry and also by their in vitro adipogenic, osteogenic and chondrogenic differentiation. This comprehensive study clearly shows that WJ is better than UCB both in terms of rapidity, yield and ease of procedure. AT and BM are autologous sources for MSC’s but the specimen collection involves cumbersome and painful procedures and an invasive approach. However being autologous, they are safe and probable candidates for therapeutic future applications.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-014-9718-z) contains supplementary material, which is available to authorized users.     

Regulation of human umbilical cord blood-derived multi-potent stem cells by autogenic osteoclast-based niche-like structure

Stem cell niches provide the micro-environment for the development of stem cells. Under our culturing regimen, a kind of osteoclast-centralized structure supports the proliferation of MSCs, derived from human cord blood, once they reside on osteoclasts. MSCs in this structure expressed Oct4 which is a marker of embryonic stem cells. Floating daughter cells of MSCs colony showed abilities to differentiate into osteocyte, adipocyte, and neuronal progenitor cells. Compared with the easy senescence of MSCs without this niche-like structure in vitro, these results suggested that osteoclasts might play an important role the development and maintenance of Umbilical cord blood (UCB)-derived MSCs and might provide a means to expand UCB-MSCs in vitro, more easily, through a stem cell niche-like structure.     

脐血间充质干细胞治疗严重性系统性红斑狼疮的临床疗效初探

目的探讨脐血间充质干细胞对严重性系统性红斑狼疮（SLE）的治疗效果。方法从健康产妇足月分娩的脐带血（UCB）中分离单个核细胞、培养制备间充质干细胞（MSCs）。MSCs培养到第三代时,取样UCB-MSCs,用流式细胞术进行性质鉴定显示：UCB-MSCs高表达CD29,CD90;不表达CD34和CD45。收集生长良好的第三代UCBMSCs。（5.0-6.0）×10^7个细胞加入100ml含1﹪人血白蛋白的生理盐水中,静脉滴注;每天1次,6-10d,一疗程;3-6个月后再用第2疗程。结果通过UCB-MSCs输注,使现有方法治疗无效的2例严重性系统性红斑狼疮患者的症状完全恢复。第1例SLE伴严重狼疮肾,狼疮脑10年,双下肢水肿,皮肤坏疽,溃疡深达骨面,剧痛难眠1年。经治医生动员她截肢保命。UCB-MSCs治疗后10d,病情明显好转,双下肢水肿几乎全部消退。治疗6个月后,11处坏疽、溃疡的疮面10个疮面基本愈合。10个月后患者的坏疽,溃疡的疮面全部愈合,能自由活动,进入正常生活。第2例SLE伴狼疮肾,狼疮脑4年,一年来贫血严重,靠每周输血维持生命。UCB-MSCs治疗二个疗程后,咳嗽,呕吐症状明显好转,浮肿和双侧胸腔积液消失,各项检查指标基本正常,不需要输血治疗。恢复休学多年的学习生活。二者均随访8年,病情稳定,没发现复发症状。结论 UCB-MSCs治疗2例严重性红斑狼疮,取得一定的疗效;这种新的技术可望成为治疗严重性系统性红斑狼疮的辅助疗法。     

Cytokine physiognomies of MSCs from varied sources confirm the regenerative commitment post-coculture with activated neutrophils

The interaction of mesenchymal stromal cells (MSCs) with paracrine signals and immunological cells, and their responses and regenerative commitment thereafter, is understudied. In the current investigation, we compared MSCs from the umbilical cord blood (UCB), dental pulp (DP), and liposuction material (LS) on their ability to respond to activated neutrophils. Cytokine profiling (interleukin-1α [IL-1α], IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], transforming growth factor-β [TGF-β]), cellular proliferation and osteogenic differentiation patterns were assessed. The results showed largely comparable cytokine profiles with higher TNF-α and IFN-γ levels in LSMSCs owing to their mature cellular phenotype. The viability and proliferation between LS/DP/UCB MSCs were comparable in the coculture group, while direct activation of MSCs with lipopolysaccharide (LPS) showed comparable proliferation with significant cell death in UCB MSCs and slightly higher cell death in the other two types of MSC. Furthermore, when MSCs post-neutrophil exposure were induced for osteogenic differentiation, though all the MSCs devoid of the sources differentiated, we observed rapid and significant turnover of DPMSCs positive of osteogenic markers rather than LS and UCB MSCs. We further observed a significant turnover of IL-1α and TGF-β at mRNA and cytokine levels, indicating the commitment of MSCs to differentiate through interacting with immunological cells or bacterial products like neutrophils or LPS, respectively. Taken together, these results suggest that MSCs have more or less similar cytokine responses devoid of their anatomical niche. They readily switch over from the cytokine responsive cell phenotype at the immunological microenvironment to differentiate and regenerate tissue in response to cellular signals.     

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 10⁶ cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.     

Mesenchymal stem cells from cryopreserved human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, but the presence of mesenchymal stem cells (MSCs) in UCB has been disputed and it remains to be validated. In this study, we examined the ability of cryopreserved UCB harvests to produce cells with characteristics of MSCs. We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of cryopreserved UCB using our culture conditions. These adherent cell populations exhibited fibroblast-like morphology and typical mesenchymal-like immunophenotypes (CD73+, CD105+, and CD166+, etc.). These cells presented the self-renewal capacity and the mesenchymal cell-lineage potential to form bone, fat, and cartilage. Moreover, they expressed mRNAs of multi-lineage genes including SDF-1, NeuroD, and VEGF-R1, suggesting that the obtained cells had the multi-differentiation capacity as bone marrow-derived MSCs. These results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and therapeutic applications.     

Expression of hyaluronan synthase genes in umbilical cord blood stem/progenitor cells

Scientific progress reveals an ever-expanding role of hyaluronan (HA) in diverse biological functions. It has become increasingly clear that HA might also be essential for certain functions of stem cells. CD133+ cells isolated from umbilical cord blood (UCB) seem to represent an alternative to CD34+ cells as a source of transplantable haematopoietic progenitor cells. The aim of this study was to investigate expression patterns of hyaluronan synthases (HAS) genes in freshly isolated and cultured UCB progenitor cells and to compare HAS mRNA levels to those found in non-progenitor cells. CD133+ stem cells were isolated from UCB using an immunomagnetic procedure. Investigation of HAS mRNA expression patterns in CD133+ and CD133- cells by RT-PCR was performed immediately after isolation as well as after cultivation towards myelomonocytic lineage. In addition, activation patterns of mitogen activated protein kinases (MAPK) were analyzed by Western blot experiments. mRNA for HAS1 is undetectable but HAS3 mRNA can be readily detected in freshly isolated CD133+ as well as in CD133- UCB cells. More importantly, our data demonstrate that mRNA for HAS2 can only be detected in CD133+ progenitor cells. In addition, while MAPK are slightly activated in CD133- UCB cells, no significant phosphorylation of MAPK could be observed in CD133+ cells, excluding a role of these kinases in the regulation of HAS2. HAS2 is expressed only in freshly isolated CD133+ cells and quickly diminishes during differentiation. Because of this, HAS2 gene expression might be suitable as a new marker for CD133+ UCB-derived stem cells.     

Isolation of three important types of stem cells from the same samples of banked umbilical cord blood

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48?h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.