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.     

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.     

<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 and osteogenic 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 its 0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the opposite results in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantly impacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

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.     

MSCs-exosomes in regeneration medicine: Current evidence and future perspectives

Exosomes, especially from mesenchymal stem cells, have attracted extensive attention in regeneration medicine. Mesenchymal stem cells derived exosomes (MSCs-exosomes) have shown anti-inflammatory, anti-oxidant, anti-apoptosis and tissue regeneration effects in a variety of tissue injury repair models. MSCs-exosomes hold many excellent properties such as low immunogenicity, biocompatibility, and targeting capability. With the in-depth study on the generation and function of exosomes, MSCs-exosomes are considered to be the bright stars in the field of regenerative medicine. However, there are still many obstacles to overcome in terms of exosomes isolation, clinical trials and safety evaluation. In this article, what we should focus on about MSCs-exosomes in regeneration medicine will be discussed.     

一种新的提取脂肪间充质干细胞的方法及对提取细胞的分离、培养及鉴定

目的：探讨建立一种新的从膨胀液中提取脂肪间充质干细胞（ADSCs）的分离方法。方法：收集含有脂肪间充质干细胞的膨胀液,然后从膨胀液中分离出脂肪间充质干细胞并进行体外培养,观察培养间充质干细胞生长状态,流式细胞术检测间充质干细胞干性标记物,细胞生长曲线比较新方法与运用传统方法分离脂肪间充质干细胞的增殖活性,多向诱导分化鉴定其向成骨,成软骨及成脂方向分化的能力。结果：成功建立了一种新的从膨胀液中提取脂肪间充质干细胞的分离方法;分离自膨胀液的间充质干细胞数量虽然低于与等体积脂肪组织来源的间充质干细胞,但细胞生长曲线分析结果表明其增殖速度快,生长至第8天时,密度基本等同于脂肪组织来源间充质干细胞。间充质干细胞表面分子标记物CD73,CD90,CD105,CD45,CD34,CD11b,CD19,HLA—DR表达测定结果显示正常,阳性细胞率与脂肪组织来源的干细胞相近。多向诱导分化结果显示从膨胀液中分离的脂肪间充质干细胞可以向成脂、成骨和成软骨三向分化。结论：新方法分离的细胞确为脂肪间充质干细胞,符合国际干细胞协会规定的定义标准。     

Microscopy analysis of bone marrow-derived osteoprogenitor cells cultured on hydrogel 3-D scaffold

Bone marrow contains progenitor cells that are able to differentiate into several mesenchymal lineages, including bone. These cells may also provide a potential therapy for bone repair. The purpose of this study was to select the osteoprogenitor cell subpopulation from bone marrow-derived mesenchymal stem cells (MSCs) and to test the ability of a hydrogel scaffold to support growth and osteogenic differentiation. MSCs isolated from rat femur bone marrow were cultured in DMEM medium supplemented with antibiotics, FCS, and L-glutamine. Osteogenic supplements (dexamethasone, sodium beta-glycerophosphate, and ascorbic acid) were added for one, two or three weeks. A selective subpopulation of osteoprogenitor cells was identified by immunohistochemistry, general morphology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Committed osteogenic cells were transferred to a 3-D hydrogel scaffold and cultured for an additional week. In standard culture, the osteoprogenitor cells formed cell clusters identified by Alizarin red S staining and by positive osteocalcin immunostaining. The number of osteoprogenitor cells, matrix synthesis, and mineralization increased gradually up to three weeks in culture. Mineral deposition in the matrix analyzed by EDS revealed the presence of calcium and phosphate ions at a Ca/P molar ratio of 1.73 in both the osteogenic cultures and the scaffold osteoprogenitor culture. Histological preparations revealed cell clusters within the hydrogel scaffold and SEM analysis revealed cell clusters attached to the scaffold surface. It is concluded that the hydrogel scaffold can support growth and differentiation of osteogenic cultures including mineralization and can potentially serve as a bone graft substitute containing committed osteoprogenitor cells.     

Immunoregulatory effects of human amniotic mesenchymal stem cells and their exosomes on human peripheral blood mononuclear cells

Background: The immunomodulatory effects of mesenchymal stem cells (MSCs) and their exosomes have been receiving increasing attention. This study investigated the immunoregulatory effects of human amniotic mesenchymal stem cells (hAMSCs) and their exosomes on phytohemagglutinin (PHA)-induced peripheral blood mononuclear cells (PBMCs). Methods: The hAMSCs used in the experiment were identified by light microscopy and flow cytometry, and the differentiation ability of the cells was determined by Oil Red O and Alizarin Red staining. The expressions of transforming growth factor (TGF)-β, indoleamine 2,3-dioxygenase (IDO), cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF), and interleukin (IL)-6 were detected by quantitative real-time polymerase chain reaction and western blotting. PBMCs, hAMSCs, and their exosomes were collected for in vitro group culture. Then the immunoregulatory ability of hAMSCs and their exosomes were analyzed by flow cytometry and Enzyme-linked immunosorbent assay. Results: The hAMSCs and exosomes were successfully extracted from the human amniotic membrane. TGF-β, IDO, COX-2, HGF, and IL-6 were significantly expressed in hAMSCs. In vitro co-culture showed that hAMSCs promoted the proliferation of Th2 cells in PHA-induced PBMCs, while hAMSCs and exosomes inhibited the secretion of TNF-α in PHA-induced PBMCs, and promoted the secretion of IL-4 and IL-10, and hAMSCs had more significant effects than exosomes. Conclusions: hAMSCs or exosomes could exert immunoregulatory effects on PHA-induced PBMCs by affecting Th2 cell proliferation and cytokine secretion.     

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.     

Controversies in therapeutic application of mesenchymal stem cell-derived secretome

Though mesenchymal stem cells (MSCs) are considered as an important pillar of regenerative medicine, their regenerative potential has been shown to be limited in several pathological conditions. The adverse properties of MSC-based cell therapy have drawn attention to the therapeutic use of MSC-derived secretome. However, MSC-originated exosomes and microvesicles can also possess a significant impact on disease development, including cancer. By interchanging secretome, MSCs can interact with tumor cells and promote mutual exchange/induction of cellular markers. In addition, enzymes secreted into and activated within exosomes can result in the acquisition of new tumor cell properties. Therefore, therapeutic applications of MSC-derived secretome require much caution.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Mesenchymal stem cells,secretome and biomaterials in <i>in-vivo</i> animal models: Regenerative medicine application in cutaneous wound healing

The treatment of nonhealing and chronic cutaneous wounds still needs a clinical advancement to be effective. Both mesenchymal stem cells (MSCs), obtained from different sources, and their secretome derived thereof (especially exosomes) can activate signaling pathways related to promotion of cell migration, vascularization, collagen deposition, and inflammatory response demonstrating prohealing, angiogenetic and anti-scarring capacities. On the other hand, biodegradable biomimetic scaffolds can facilitate endogenous cell attachment and proliferation as well as extracellular matrix production. In this Review, we revise the complex composites made by biomimetic scaffolds, mainly hydrogels, and MSC-derived exosomes constructed for cutaneous wound healing. Studies demonstrate that there exists a synergistic action of scaffolds with encapsulated exosomes, displaying a sustained release profiles to facilitate longlasting healing effects. It can be envisioned that dressings made by biomimetic hydrogels and MSC-derived exosomes will be clinically applied in the near future for the effective treatment of nonhealing and chronic wounds.     

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/heroine kinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEM medium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the following groups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylation inhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342 and 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 elevated levels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, the H9c2 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 was lower 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), but decreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secrete multiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which the activation of Akt phosphorylation is involved to achieve the protective effect.     

Adipose-derived mesenchymal stem/stromal cells: from the lab bench to the basic concepts for clinical translation

In the last years, much work has shown that the most effective repair system of the body is represented by stem cells, which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal ability, which also have the potential to transform themselves into various cell types through differentiation.All tissues that form the body contain many different types of somatic cells, along with stem cells that are called ‘mesenchymal stem (or stromal) cells’ (MSC). In certain circumstances, some of these MSC migrate to injured tissues to replace dead cells or to undergo differentiation to repair it.The discovery of MSC has been an important step in regenerative medicine because of their high versatility. Moreover, the finding of a method to isolate MSC from adipose tissue, so called ‘adipose-derived mesenchymal stem cells’ (ASC), which share similar differentiation capabilities and isolation yield that is greater than other MSC, and less bioethical concerns compared to embryonic stem cells, have created self-praised publicity to procure almost any treatment with them. Here, we review the current techniques for isolation, culture and differentiation of human ASC (hASC), and describe them in detail. We also compile some advantages of the hASC over other stem cells, and provide some concepts that could help finding strategies to promote their therapeutic efficiency.     

Tissue engineering of the synovial joint: the role of cell density

The ultimate goal in the tissue engineering of the synovial joint is to fabricate biologically derived analogues that can replace severely degenerated or traumatized synovial joint components. A number of challenges must be addressed before reaching this ultimate goal. In this report, the relevance of cell seeding density in the synthesis of chondrogenic and osteogenic matrices from human mesenchymal stem cells is explored. Human mesenchymal stem cells (hMSCs) were differentiated into chondrogenic cells and osteogenic cells ex vivo and encapsulated in poly(ethylene glycol) diacrylate (PEGDA) hydrogel at densities of 5 x 106 cells/ml, 40 x 10(6) cells/ml, and 80 x 10(6) cells/ml, in addition to a cell-free poly(ethylene glycol) (PEG) control group (0 x 10(6) cells/ml). Cell-seeded or cell-free PEG constructs were separately incubated in vitro for 4 weeks or implanted in vivo in the dorsum of immunodeficient rats for 4 weeks. In-vitro data demonstrated that hMSC-derived chondrocytes or hMSC-derived osteoblasts maintained their lineages per Safranin O and von Kossa staining after incubation for 4 weeks. The general pattern of initial cell seeding densities of 5 x 10(6) cells/ml, 40 x 10(6) cells/ml, and 80 x 10(6) cells/ml were preserved following in-vitro cultivation. Similarly, in-vivo data revealed that hMSC-derived chondrocytes and hMSC-derived osteoblasts maintained their respective lineages and the pattern of cell-seeding densities. An attempt was made to fabricate a composite construct with PEGDA hydrogel and polycaprolactone (PCL) with designed internal porosity for an osteochondral graft. Various cell-seeding densities as delineated in this report can be realized in the composite PEG-PCL graft. The findings demonstrate that cell-seeding density is likely a key parameter to consider in tissue-engineering design. The source of cells can either be transplanted cells or internally recruited cells.