Phenotypic,morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography–mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44⁺, CD105^low, CD90^low, exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.     

Cell-surface ultrastructural changes during the in vitro neuron-like differentiation of rat bone marrow-derived mesenchymal stem cells

The neuron-like differentiation of bone marrow-derived mesenchymal stem cells (BMMSCs) has been extensively studied. However, the alternations of the cell-surface ultrastructures and the membrane tension/reservoir of the cells during this differentiation process are poorly understood. Therefore, atomic force microscopy (AFM) was utilized in this study to observe the cell-surface ultrastructural changes among rat bone marrow-derived mesenchymal stem cells (rBMMSCs), partially differentiated cells, and fully differentiated neuron-like cells. By analyzing the stiffness of plasma membranes, lamellipodial extensions, average heights of small membrane protrusions and relatively larger uplifted structures, and peak-peak spacing among protrusions and/or uplifted structures, we found that the membrane reservoir may potentially decrease upon the differentiation from rBMMSCs to partially differentiated cells and to fully differentiated neuron-like cells. The results may help to better understanding the membrane tension of various types of cells and related biological processes, such as membrane traffic, cell adhesion, motility, differentiation, among others. The data also implies that AFM may be a useful tool for evaluating membrane reservoir by imaging cell-surface ultrastructures.     

Astragaloside IV improves melanocyte differentiation from mouse bone marrow mesenchymal stem cells

Vitiligo results in an autoimmune disorder destructing skin pigment cells, melanocytes (Mcs). This study aimedto investigate whether Astragaloside IV (AIV) could efficiently induce differentiation of bone marrow mesenchymal stemcells (BMMSCs) into Mcs. BMMSCs were induced and differentiated into Mcs with 0.1, 0.2, and 0.4 mg/L AIV during150-day. Morphologic changes of differentiated cells were observed. Levels of some melanocytic specific genes (TRP-1,TRP-2, MART-1, Mitf) were measured with quantitative polymerase chain reaction (qPCR) at 90, 120, and 150 daysof induction. After 90-day induction, the differentiated cells with 0.4 mg/L AIV demonstrated the typical morphologyof Mcs, positive 3,4 dihydroxyphenylalanine staining, and positive staining of TRP-1, TRP-2, MART-1, and Mitf.After 90- and 120- days’ induction with 0.4 mg/L AIV, TRP-1 expression was significantly elevated (p < 0.01), andTRP-2 expression was significantly increased in 0.4 mg/L AIV-treated group compared to negative control (p < 0.01),0.1 mg/L (p < 0.01), and 0.2 mg/L (p < 0.01) AIV-treated groups. Moreover, MART-1 expression was significantlyup-regulated in 0.4 mg/L AIV-treated group compared to negative control, but without difference compared to 0.1mg/L (p > 0.05) and 0.2 mg/L (p > 0.05) AIV-treated groups. During 90 to 150- day induction, there were nosignificant differences for Mitf levels between AIV-treated groups and negative control (p > 0.05). In conclusion,90-day induction with 0.4 mg/L AIV up-regulated TRP-1, TRP-2, and MART-1 expression, indicating that AIV canefficiently induce Mcs differentiation from BMMSCs. These results provide experimental and theoretic evidence forAIV application in clinical vitiligo repigmentation treatment.     

Transplantation of BMP-7 gene-transfected bone marrow mesenchymal stem cells for the treatment of spinal cord injury in rats

Background: Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system, and there is currently no effective treatment for SCI because of its complicated pathophysiology. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation abilities. Our study aims to explore the effects of bone morphogenetic protein 7 (BMP-7)-modified BMSCs transplantation on the repair of SCI in rats. Methods: In this study, a rat spinal cord injury model was established with the modified Allen method. Then, BMSCs transfected with the BMP7 gene were transplanted to treat the spinal cord injury in rats. Forty Sprague-Dawley rats were randomly divided into the sham operation group (sham group), spinal cord injury group (model group), BMSC treatment group (BMSC group) and LV-BMP7-BMSC treatment group (LV-BMP7-BMSC group). The Basso, Beattie, and Bresnahan (BBB) score was used to evaluate the recovery of hindlimb function in the rats. The levels of neurofilament protein NF-200 (NF-200) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence, RT-PCR and Western blotting. Results: At 14 d, 21 d, and 28 d after treatment, the BBB score of the rats in the LV-BMP7-BMSC group was higher than that of the rats in the model group and BMSC group. The results showed that NF-200 was expressed at the local spinal cord injury site. Compared with that of the sham group, the NF-200 expression level of the BMSC group and LV-BMP7-BMSC group was increased (P < 0.05). The results showed that the mRNA expression levels of NF-200 in the spinal cord tissue of the BMSC group and LV-BMP7-BMSC group were increased compared with those of the sham group (P < 0.05). The western blotting results further confirmed the PCR results. Conclusion: BMP-7 gene-modified BMSC transplantation can promote the repair of spinal cord functions after SCI in rats.     

Immunophenotypic evaluation,and physiological and laboratory correlations of hematopoietic stem cells from umbilical cord blood

The use of umbilical cord blood stem cells is an efficient alternative for the transplantation of hematopoietic progenitor cells. A number of factors can influence the volume and amount of CD34⁺ cells, which are considered as immature and capable of proliferation. Quantification of CD34⁺ cells, evaluation of CD38 and c-kit molecules on these cells, as well as correlations of such factors as maternal age, gestational age, newborn sex and weight, umbilical cord length, placental weight with increased volume and concentration of immature cells, among others, were performed in 70 blood samples from term newborns. The mean volume of umbilical cord blood collected was 53.8±33.6 mL, where 30.96±18.9 CD34⁺/µL UCB cells were found, of which 16.66±8.32% were CD34⁺ CD38- cells, and 47.23±24.0% were CD34⁺ CD117^- cells. Newborn weight and placental weight were positively correlated with increased volume of collected UCB. The volume of collected blood was found to affect the absolute count of CD34⁺ cells and the relative value of these among total nucleated cells, as well as the percentage of CD34⁺CD117⁺ and CD34⁺CD117^- cells. CD34⁺ cells were positively correlated with leukocytes, and gestational age was negatively correlated with the number of CD34⁺ cells. Our results confirm the importance of the accurate quantification of CD34⁺ cells and their subsets, and that many factors may be related to the higher number of hematopoietic stem cells, which are crucial for successful transplantation.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs(miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic andosteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified.Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold inMDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenicdifferentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the oppositeresults in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantlyimpacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation whileweakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

Identification of tumorigenic risk genes in human placenta-derived mesenchymal stem cells treated with 3-methylcholanthrene

Mesenchymal stem cells (MSCs) capable of tumour topotaxis have been served as cellular vehicles to deliveranti-tumour agents. As cellular components of the tumour microenvironment, MSCs also affect tumour progression.However, the tumour transformation-related genes of MSCs remain unclear since either tumorigenic or tumoursuppressor effects within these cells have been researched. Hence, we aimed to identify potential biomarkers indicativeof tumorigenic risk by RNA-seq analysis of human placenta tissue-derived MSCs (hPTMSCs) exposed to thecarcinogenic agent, 3-methylcholanthrene (3-MC). Twenty-nine tumour transformation-related genes and threepluripotency-related genes were appraised as differentially expressed genes (DEGs) in hPTMSCs. Overexpression ofsfrp1 led to reduced cell viability, migration, and colony formation in A549. In contrast, the overexpression of ptgs2exerted the opposite effect. These results indicate that A549 cells with high ptgs2 expression but low sfrp1 expressionmay have a more potential tumorigenic capacity. Taken together, this study suggests that ptgs2 and sfrp1 may betumorigenic risk genes.     

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells (iNSCs) transplantation is a potential treatment of neurodegenerationdiseases. However, whether the reprogrammed cells have the same characterizations as human fetal neural stem cellsneeds further exploration. Here we isolated human fetal neural stem cells from aborted 12-week fetal brains andcompared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression, proliferationability, differentiation capacity, and the responses to tumor necrosis factor-α. We found that iNSCs and NSCs bothexpressed neural stem cell markers Nestin, SOX1, and SOX2. However, only iNSCs can be patterned into dopaminergicneurons and motor neurons. Furthermore, both iNSCs and NSCs can differentiate into oligodendrocyte progenitorcells. In addition, a low dose of tumor necrosis factor-α did not inhibit the proliferation and differentiation of iNSCsand NSCs. In conclusion, iNSCs have properties similar to, and even better than, fetal neural stem cells and may besuitable for disease modeling and transplantation.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Ultrastructural analysis of human bone marrow mesenchymal stem cells during in vitro osteogenesis and chondrogenesis

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroinekinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEMmedium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the followinggroups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylationinhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD)for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevatedlevels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, theH9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group,while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group waslower than the hypoxia group (12.34 ± 2.00% vs. 21.73 ± 2.58%; p < 0.05), while the LY group was significantly higher(22.54 ± 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (p < 0.05), butdecreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secretemultiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which theactivation of Akt phosphorylation is involved to achieve the protective effect.     

M1 macrophage-derived exosomes moderate the differentiation of bone marrow mesenchymal stem cells

Differentiated macrophages have been proven to participate in the development of mesenchymal stem cells in different tissues. However, the regulatory processes remain obscure. Exosomes, which are key secretions of macrophages, have attracted increasing attention. Therefore, macrophage-derived exosomes may modulate the development of Bone marrow mesenchymal stem cells (BMMSCs). Different culture conditions were used to induce M1 polarization in THP1 cells. Subsequently, exosomes derived from unpolarized (M0) and polarized (M1) macrophages were isolated, BMMSCs were cultured with normal complete medium or inductive medium supplemented with M0 or M1 derived exosomes, and the osteogenic capacity of the BMMSCs was measured and analyzed. Finally, molecular mechanism associated with Akt and RUNX2 was investigated. Alizarin red staining and WB experiments showed that M1 macrophages could promote the osteogenic differentiation of BMMSCs better than M0 macrophages. Then, exosomes derived from M0 and M1 macrophages were successfully isolated and analyzed by electron microscopy and WB experiments. We concluded that media containing M1-derived exosomes promoted the osteogenic differentiation of BMMSCs better than media containing M0-derived exosomes. In addition, M1-derived exosomes could activate Akt and increase RUNX2 levels to promote osteogenesis. Our data demonstrated that exosomes derived from M1 macrophages induced osteogenesis by activating Akt and increasing RUNX2 level.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.