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.     

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.     

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.     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.     

Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells

In vitro cellular proliferation and the ability to undergo multilineage differentiation make bone marrow-derived multipotent stromal cells (MSCs) potentially useful for clinical applications. Several methods have been described to isolate a homogenous bone marrow-derived MSCs population; however, none has been proven most effective, mainly due to their effects on proliferation and differentiation capability of the isolated cells. It is hypothesized that our newly established total cell pooling method may provide a better alternative as compared to the standard isolation method (density gradient centrifugation method). For the total cell pooling method, MSCs were isolated from rabbit bone marrow and were subsequently cultured in the growth medium without further separation as in the standard isolation method. The total cell pooling method was 65 min faster than the standard isolation method in completing cell isolation. Nevertheless, both methods did not differ significantly in the number of primary viable cells and population doubling time in the cultures (p?>?0.05). The isolated cells from both methods expressed CD29 and CD44 markers, but not CD45 markers. Furthermore, they displayed multilineage differentiation characteristics of chondroblasts, osteoblasts, and adipocytes. In conclusion, both methods provide similar efficiency in the isolation of rabbit bone marrow-derived MSCs; however, the total cell pooling method is technically simpler and more cost effective than the standard isolation method.     

Comparative evaluation of in vivo osteogenic differentiation of fetal and adult mesenchymal stem cell in rat critical-sized femoral defect model

Mesenchymal stem cells (MSCs) can be obtained from various sources. MSCs from different origins appear to have different preferences for differentiation. In this study, we have compared the in vivo osteogenic potential of adult MSCs from adipose tissue (AT) and bone marrow (BM) with fetal MSCs from umbilical cord (UC) and umbilical cord blood (UCB) by using a rat critical-sized femoral defect model. We have also sought to determine whether pretreatment with an osteogenic medium promotes osteogenesis in MSCs. Study groups were divided as follows: (1) defect only, (2) scaffold only, (3) AT MSCs in scaffolds, (4) BM MSCs in scaffolds, (5) UC MSCs in scaffolds and (6) UCB MSCs in scaffolds. Groups with MSCs were further divided with respect to their pretreatment. At 12 weeks after surgery, in vivo osteogenesis was measured radiographically and by micro-computed tomography (CT). Based on quantitative assessment by micro-CT, no significant difference of the mean bone volume fraction value (BV/TV) was seen between adult MSCs (AT and BM MSCs) and fetal MSCs (UC and UCB MSCs). The mean BV/TVs were significantly higher in non-pretreated BM MSC (14.2±1.4%) and UCB MSC (14.0±1.2%) and pretreated UC MSC (14.8±2.0%) than in those with the scaffold only (11.3±1.3%; P<0.05). In addition, AT (from 10.4±1.2% to 13.1±2.2%) and UC (from 10.3±0.7% to 14.8±2.0%) MSCs from solid tissues showed a significant increase in the mean BV/TV with pretreatment (P<0.05). In contrast, BM MSC (from 14.2±1.4% to 10.9±1.2%) and UCB MSC (from 14.0±1.2% to 11.6±1.0%) from non-solid tissues showed a significant decrease with pretreatment (P<0.05).     

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     

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast‐like, and the population doubling time (DT) significantly increased with passage number. For AF‐ and AM‐MSCs, cell viability did not change with passages. In UCM‐MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM‐MSCs expressed embryonic and MSC markers, such as Oct‐4 CD44, CD184, and CD29, whereas AF‐MSCs expressed Oct‐4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA‐DRA1 and DLA‐79) were expressed only in AF‐MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs. Mol. Reprod. Dev. 78:361–373, 2011. © 2011 Wiley‐Liss, Inc.     

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.     

Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods

Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton’s jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10? cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90–99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13–40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.     

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.     

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.     

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.     

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer–irrigator–aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7–10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.     

Mesenchymal stroma cells in peritoneal dialysis effluents from patients

Mesenchymal stroma cells (MSCs) have potential as an emerging cell therapy for treating many different diseases, but discovery of the practical sources of MSCs is needed for the large-scale clinical application of this therapy. This study was to identify MSCs in peritoneal dialysis (PD) effluents that were discarded after PD. The effluents were collected from patients who were on the dialysis for less than 1 month. Adherent cells from the effluents were isolated by incubation in serum-containing medium in plastic culture dishes. Cell surface markers were determined by a flow cytometric analysis, and the in vitro differentiation to chondrocytes, osteocytes or adipocytes was confirmed by staining with a specific dye. After four passages, these isolated cells displayed the typical morphology of mesenchymal cells in traditional 2-D cultures, and were grown to form spherical colonies in 3-D collagen cultures. Flow cytometric analysis revealed that the unsorted cells from all of seven patient samples showed robust expression of typical mesenchymal marker CD29, CD44, CD73, CD90 and CD166, and the absence of CD34, CD79a, CD105, CD271, SSEA-4, Stro-1 and HLA-DR. In differentiation assays, these cells were induced in vitro to chondrocytes, osteocytes or adipocytes. In conclusion, this preliminary study suggests the presence of MSCs in the “discarded” PD effluents. Further characterization of the phenotypes of these MSCs and evaluation of their therapeutic potential, particularly for the prevention of PD failure, are needed.     

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.     

CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood

Background aimsUmbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.MethodsWe assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133⁺ subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.ResultsThe mononuclear cell fraction expressed 0.53 ± 0.06% CD133. The corresponding value for CD34 was 1.64 ± 0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93 ± 1.25% CD34 while, after the corresponding process, CD133⁺ expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133⁺ yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).ConclusionsCD34 isolation by the immunomagnetic method results in highly pure CD34⁺ population, while the efficient and reproducible yield of a pure CD133⁺ population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133⁺ cells.     

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.