Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells

Placenta-derived stem cells (PDSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal and differentiation and their immunomodulatory properties. Although many studies have characterized various PDSCs biologically, the properties of the self-renewal and differentiation potential among PDSCs have not yet been directly compared. We consider the characterization of chorionic-plate-derived mesenchymal stem cells (CP-MSCs) and Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) among various PDSCs and the assessment of their differentiation potential to be important for future studies into the applicability and effectiveness of PDSCs in cell therapy. In the present study, the capacities for self-renewal and multipotent differentiation of CP-MSCs and WJ-MSC isolated from normal term placentas were compared. CP-MSCs and WJ-MSCs expressed mRNAs for the pluripotent stem cell markers Oct-4, Nanog, and Sox-2. Additionally, HLA-G for immunomodulatory effects was found to be expressed at both the mRNA and protein levels in both cell types. The CP-MSCs and WJ-MSCs also had the capacities to differentiate into cells of mesodermal (adipogenic and osteogenic) and endodermal (hepatogenic) lineages. Expression of adipogenesis-related genes was higher in CP-MSCs than in WJ-MSCs, whereas WJ-MSCs accumulated more mineralized matrix than CP-MSCs. The WJ-MSCs expressed more of CYP3A4 mRNA, a marker for mature hepatocytes, than CP-MSCs. Thus, we propose that CP-MSCs and WJ-MSCs are useful sources of cells for appropriate clinical applications in the treatment of various degenerative diseases.     

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.     

Are mesenchymal stem cells major sources of safe signals in immune system?

Numerous reports have shown that mesenchymal stem cells (MSCs) are implicated in immuno-regulation. Several factors expressed from MSCs, especially indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2), are of importance in immuno-regulation on immune cells. In current minireview, we provided evidences to support a novel notion that MSCs may be a major source of "safe signals" in the immune system to balance "dangerous signals" based on a well accepted theory of "danger model". Furthermore, MSCs are of lifecycle characterized by age-and diseased-related changes, such as decreased growth rate, increased senescence, and altered morphology. Thus, defected and abnormal MSCs are implicated in auto-immune diseases, such as systemic lupus erythematosus (SLE). Clinically, it is important to determine clinical benefits and sides effects of cell therapies using autologous self-MSCs or healthy allogeneic MSCs in treatment of autoimmune diseases.     

Study of Wnt2 secreted by A-549 cells in paracrine activation of β-catenin in co-cultured mesenchymal stem cells

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.     

Effect of TNFα on osteoblastogenesis from mesenchymal stem cells

Background

Bone destruction and osteoporosis are accelerated in chronic inflammatory diseases, such as rheumatoid arthritis (RA) and periodontitis, in which many studies have shown the proinflammatory cytokines, especially TNFα, play an important role; TNFα causes osteoclast-induced bone destruction as well as the inhibition of osteoblastogenesis.

Scope of review

Here we review our current understanding of the mechanism of the effect of TNFα on osteoblastogenesis from mesenchymal stem cells (MSCs). We also highlight the function of MSC in the pathogenesis of autoimmune diseases.

Major conclusions

Many studies have revealed that TNFα inhibits osteoblastogenesis through several mechanisms. On the other hand, it has been also reported that TNFα promotes osteoblastogenesis. These discrepancies may depend on the cellular types, the model animals, and the timing and duration of TNFα administration.

General significance

A full understanding of the role and function of TNFα on osteoblastogenesis from MSC may lead to targeted new therapies for chronic inflammation diseases, such as RA and periodontitis.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Brain mesenchymal stem cells:The other stem cells of the brain?

Multipotent mesenchymal stromal cells(MSC),have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation.The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair.However,some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist.In brain,perivascular MSCs like pericytes and adventitial cells,could constitute another stem cell population distinct to the neural stem cell pool.The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes,the demonstration of neural biomarkers expression,electrophysiological recordings,and the absence of cell fusion.The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells.It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.     

Human mesenchymal stem cells-like cells as cellular vehicles for delivery of immunotoxin in vitro

Human mesenchymal stem cells-like cells (hMSCs-like cells) were used as a tumor treatment platform for the systemic delivery of immunotoxin genes. VEGF165-PE38 recombinant immunotoxin served as the model system. hMSCs-like cells were isolated, expanded, and electroporated with the pIRES2-VEGF165PE38-EGFP plasmid. RT-PCR and ELISA were used to confirm the expression of VEGF165-PE38 in the transfected hMSCs-like cells. These cells released 1390 ± 137 pg VEGF165-PE38/10⁴cells over 48 h into the culture medium and the supernatant was capable of selectively killing human umbilical vein endothelial cells (HUVECs) and increasing apoptosis in these cells. In contrast, RPMI8226 was not inhibited by identical supernatants. Thus, these results lay the foundation for further studies on the potential role of hMSCs-like cells as a targeted therapeutic delivery vehicle for immunotoxins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O₂ concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 10⁵ cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O₂ uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.     

In vitro chondrogenesis of Wharton’s jelly mesenchymal stem cells in hyaluronic acid-based hydrogels

Background

In this study, we evaluated the usefulness of two commercially available hyaluronic acid-based hydrogels, HyStem and HyStem-C, for the cultivation of Wharton’s jelly mesenchymal stem cells (WJ-MSCs) and their differentiation towards chondrocytes.

Methods

The WJ-MSCs were isolated from umbilical cord Wharton’s jelly using the explant method and their immunophenotype was evaluated via flow cytometry analysis. According to the criteria established by the International Society for Cellular Therapy, they were true MSCs. We assessed the ability of the WJ-MSCs and chondrocytes to grow in three-dimensional hydrogels and their metabolic activity. Chondrogenesis of WJ-MSCs in the hydrogels was determined using alcian blue and safranin O staining and real-time PCR evaluation of gene expression in the extracellular matrixes: collagen type I, II, III and aggrecan.

Results

Chondrocytes and WJ-MSCs cultured in the HyStem and HyStem-C hydrogels adopted spherical shapes, which are characteristic for encapsulated cells. The average viability of the WJ-MSCs and chondrocytes in the HyStem hydrogels was approximately 67 % when compared with the viability in 2D culture. Alcian blue and safranin O staining revealed intensive production of proteoglycans by the cells in the HyStem hydrogels. Increased expression of collagen type II and aggrecan in the WJ-MSCs cultured in the HyStem hydrogel in the presence of chondrogenic medium showed that under these conditions, the cells have a high capacity to differentiate towards chondrocytes. The relatively high viability of WJ-MSCs and chondrocytes in both HyStem hydrogels suggests the possibility of their use for chondrogenesis.

Conlusions

The results indicate that WJ-MSCs have some degree of chondrogenic potential in HyStem and HyStem-C hydrogels, showing promise for the engineering of damaged articular cartilage.

     

Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats

Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs’ therapeutic efficiency in HI and the promotion of the cells’ directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future.     

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, na?ve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.     

Wnt/β-catenin signaling regulates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into neuron-like cells, but the precise mechanisms controlling this process are unclear. Using neuron-specific enolase (NSE) and nestin as neuronal markers, we examined the role of Wnt/β-catenin signaling in MSC neuronal differentiation in present study. The results indicated that the expression of β-catenin increased markedly during the neuronal differentiation of MSCs. Blocking Wnt signaling by treating MSCs with β-catenin siRNA could decrease the differentiation of MSCs into neuron-like cells and up-regulation of Wnt signaling by treating MSCs with Wnt-3a could promote neuronal differentiation of MSCs. Above results suggest that Wnt/β-catenin signaling may play a pivotal role in neuronal differentiation of MSCs. Our data broaden the knowledge of molecular mechanisms involved in the neuronal differentiation of MSCs and provide a potential target for directing differentiation of MSCs for clinical application.     

Cardiomyocyte-like cells differentiation from non β-catenin expression mesenchymal stem cells

Recent studies have shown that block wnt/β-catenin signaling pathway is integrant for cardiomyocytes differentiation from bone marrow mesenchymal stem cells (MSCs). By transducing the MSCs with lentivirus which contain β-catenin interference RNA, we screened out the non β-catenin expression clone. In the establishment of knockdown β-catenin in MSCs, we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (salB), and cardiomyocytes lysis medium (CLM) in inducing MSCs to differentiate into cardiomyocyte-like cells. A method for culturing MSCs and cardiomyocytes was established. Purified MSCs were investigated by flow cytometry. The MSCs were positive for CD90 and CD29, but negative for CD34 and CD45. Meanwhile, the cardiomyocytes contracted spontaneously after 24 h of seeding into the plates. The fourth-passage non-β-catenin expression MSCs were divided into eight groups: control group, 5-aza, salB, CLM, 5-aza + salB, 5-aza + CLM, salB + CLM, and 5-aza + salB + CLM. The gene and protein expression of cTnT, α-actin, β-myosin, β-catenin, and GSK-3β were detected by quantitative real-time PCR and Western blotting. Our results showed that cTnT expression in 5-aza + salB + CLM group was ninefold higher than in the control group in the non-β-catenin MSCs model, implying that cardiomyocytes differentiation from MSCs is an extremely complicated process and it is necessary to consider the internal and external environmental conditions, such as suitable pharmaceutical inducers, cardiomyocytes microenvironments, inhibition of the negative signaling pathway and so on.     

Perinatal sources of mesenchymal stem cells: Wharton’s jelly,amnion and chorion

Recently, stem cell biology has become an interesting topic, especially in the context of treating diseases and injuries using transplantation therapy. Several varieties of human stem cells have been isolated and identified in vivo and in vitro. Ideally, stem cells for regenerative medical application should be found in abundant quantities, harvestable in a minimally invasive procedure, then safely and effectively transplanted to either an autologous or allogenic host. The two main groups of stem cells, embryonic stem cells and adult stem cells, have been expanded to include perinatal stem cells. Mesenchymal stem cells from perinatal tissue may be particularly useful in the clinic for autologous transplantation for fetuses and newborns, and after banking in later stages of life, as well as for in utero transplantation in case of genetic disorders.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Involvement of TNF-α in differential gene expression pattern of CXCR4 on human marrow-derived mesenchymal stem cells

Cell therapy and tissue repair are used in a variety of diseases including tissue and organ transplantation, autoimmune diseases and cancers. Now mesenchymal stem cells (MSCs) are an attractive and promising source for cell-based therapy according to their individual characteristics. Soluble factors which are able to induce MSCs migration have a vital role in cell engraftment and tissue regeneration. Tumor necrosis factor α (TNF-α) is a major cytokine present in damaged tissues. We have investigated the pattern of gene expression of chemokine receptor CXCR4 in nine groups of human bone marrow-derived MSCs stimulated with TNF-α in different dose and time manner. Comparison of TNF-α treated with untreated MSCs revealed the highest expression level of CXCR4 after treatment with 1, and 10 ng/ml of TNF-α in 24 h, and the production of CXCR4 mRNA was regulated up to 216 and 512 fold, respectively. Our results demonstrated the differential gene expression pattern of chemokine receptor CXCR4 in human marrow-derived MSCs stimulated with inflammatory cytokine TNF-α. These findings suggest that in vitro control of both dose and time factors may be important in stem cell migration capacity, and perhaps in future-stem cell transplantation therapies.     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Differentiation of mesenchymal stem cells into gonad and adrenal steroidogenic cells

Hormone replacement therapy is necessary for patients with adrenal and gonadal failure.Steroid hormone treatment is also employed in aging people for sex hormone deficiency.These patients undergo such therapies,which have associated risks,for their entire life.Stem cells represent an innovative tool for tissue regeneration and the possibility of solving these problems.Among various stem cell types,mesenchymal stem cells have the potential to differentiate into steroidogenic cells both in vivo and in vitro.In particular,they can effectively be differentiated into steroidogenic cells by expressing nuclear receptor 5A subfamily proteins(steroidogenic factor-1 and liver receptor homolog-1)with the aid of cAMP.This approach will provide a source of cells for future regenerative medicine for the treatment of diseases caused by steroidogenesis deficiencies.It can also represent a useful tool for studying the molecular mechanisms of steroidogenesis and its related diseases.