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.     

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。     

Chondrogenic differentiation of human adipose mesenchimal stem cells: Influence of a biomimetic gelatin genipin crosslinked porous scaffold

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928–934, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Integrated Membrane Proteomic Strategies Revealed Cell Surface Signature during Human Embryonic Stem Cell Differentiation

In stem cell research, cell surface markers are extensively used for stem cell classification, monitoring the differentiation stages as well as purification for their use in regenerative medicine. Quantitative membrane proteomic approaches will provide an in-depth view of the stage- and lineage-specific expression which potentially enhances our understanding on the underlying mechanisms of stem cell differentiation, as well as the opportunity towards isolation of homogenous primary stem cell population. However, the analysis of membrane proteome and its highly heterogeneous glycosylation is experimentally challenging because of their hydrophobic nature and low abundance, which seriously complicate their solubilization, sample handling, separation, and mass spectrometric analysis. In attempt to search for novel stem cell surface markers and differentiation regulators, we have applied a subglobal proteomic approach and glycoproteomic profiling to define a “membrane proteomic profile” of human embryonic stem (hES) cells and 16-day differentiated embryoid body (EB) outgrowth. Using our recently reported gel-assisted digestion and iTRAQ labeling approach, 3842 proteins were identified (p<0.05) and 2783 proteins were quantified with 2 peptides. By labeling strategy with alkynyl sugar derivatives, the preliminary results In glycoproteomic analysis identified 350 glycopeptides (p<0.05) and quantified in 212 glycoproteins. By combining the quantitative information in protein expression and N-glycosylated peptides, the site-specific glycosylation degree of peptides can be confidently determined on a proteome scale. Our study revealed the dramatic change in expression as well as sialylated N-glycosylation on cell surface glycoproteome during stem cell differentiation. Interestingly, the proteomic data revealed that some cell surface markers, previously discovered by gene expression array, have unaltered expression during stem cell differentiation, which may be due to differences in protein turnover and regulation of the abundance of cellular mRNA and proteins. Mapping of these differentially expressed glycoproteins and membrane proteins in multiple cellular pathways related to cell differentiation, proliferation and cell development suggests that not only the protein markers but also the site and degree of glycosylation have distinct pattern or function in the complex process during stem cell differentiation.     

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.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

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.     

<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.     

A novel nine gene signature integrates stemness characteristics associated with prognosis in hepatocellular carcinoma

Cancer stem cells (CSCs) are heterogeneous with self-renewal and differentiation ability. The mRNA expression-based stemness index (mRNAsi) described the similarity between tumor cells and CSCs, which is positively associated with the poor prognosis of cancer patients. However, the key prognostic genes related to mRNAsi in hepatocellular carcinoma (HCC) remains unclear. A 9-gene signature related to mRNAsi and HCC prognosis including PSMG3, SNRPD1, DTYMK, PIGU, NME1, TXNL4A, IPO4, PES1, and REXO4 was obtained. High expression of this signature indicates poor prognosis of HCC. PIGU was an independent prognostic factor of HCC, which was significantly associated with progression of HCC. Among them, DTYMK and NME1 enriched in pyrimidine metabolism, SNRPD1 and TXNL4A enriched in spliceosome and PIGU enriched in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis pathways. High levels of IPO4, NME1, PES1, PIGU and SNRPD1 were closely associated with metastasis of HCC, and low levels of IPO4, PIGU and REOX4 were significantly associated with sorafenib resistance of HCC. High expression of the 9-gene signature was negatively correlated with the stromal cell infiltration, and positively correlated with specific immune subtypes-related to angiogenesis, M1/M2 macrophage polarization, and M2 response. The 9-gene signature was negatively correlated with the stroma, and SNRPD1 and TXNL4 were positively correlated with immune infiltrate. NME1 was negatively correlated with tumor purity. Therefore, a 9-gene signature related to mRNAsi and poor prognosis in HCC were identified, which can be used as biomarkers for the diagnosis of HCC and functional mechanism exploration of CSCs in HCC. These genes such as IPO4 and PIGU might drive the transition of tumor cells into CSCs which possibly controls the balance between metastasis and drug resistance in HCC. The challenge on balance between metastasis and drug resistance for tumor therapy was firstly reported by the present study.     

Wnt3a-induced ST2 decellularized matrix ornamented PCL scaffold for bone tissue engineering

The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering. Wnt3a activates the classic Wnt/β-catenin signaling pathway which effects bone growth and development by the accumulation of β-catenin in the nucleus. In this study, we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix (Wnt3a-ST2-dCM-PCL) and ST2 decellularized matrix (ST2-dCM-PCL) by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized >90% DNA while preserved most protein. Compared to ST2-dCM-PCL, Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation, osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase (Alp), Runx2, type I collagen (Col 1) and osteocalcin (Ocn) mRNA expression. After 14 days of osteogenic induction, Wnt3a-ST2-dCM-PCL promoted ST2 mineralization. These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’ osteoinductivity and osteoconductivity.     

Stem cells for tissue engineering of articular cartilage

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.     

New insights into the cellular makeup and progenitor potential of palatal connective tissues

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco‐gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de‐epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de‐epithelialised MSCs (deMSCs) and re‐entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony‐forming unit‐ fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA‐DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA?, CD31?) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,—in the pericapillary regions and in remote regions of the lamina propria‐ and pericytes—surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision‐making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.