Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Necrotic and apoptotic cells serve as nuclei for calcification on osteoblastic differentiation of human mesenchymal stem cells in vitro

A close relationship between cell death and pathological calcification has recently been reported, such as vascular calcification in atherosclerosis. However, the roles of cell death in calcification by osteoblast lineage have not been elucidated in detail. In this study, we investigated whether cell death is involved in the calcification on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hMSC) under osteogenic culture in vitro. Apoptosis and necrosis occurred in an osteogenic culture of hMSC, and cell death preceded calcification. The generation of intracellular reactive oxygen species, chromatin condensation and fragmentation, and caspase‐3 activation increased in this culture. A pan‐caspase inhibitor (Z‐VAD‐FMK) and anti‐oxidants (Tiron and n ‐acetylcysteine) inhibited osteogenic culture‐induced cell death and calcification. Furthermore, calcification was significantly promoted by the addition of necrotic dead cells or its membrane fraction. Spontaneously dead cells by osteogenic culture and exogenously added necrotic cells were surrounded by calcium deposits. Induction of localized cell death by photodynamic treatment in the osteogenic culture resulted in co‐localized calcification. These findings show that necrotic and apoptotic cell deaths were induced in an osteogenic culture of hMSC and indicated that both necrotic and apoptotic cells of osteoblast lineage served as nuclei for calcification on osteoblastic differentiation of hMSC in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140]     

干细胞向胰岛样细胞诱导分化的研究进展

目前,糖尿病患病率呈现上升趋势,它已成为严重危害人类健康的疾病之一。胰岛细胞移植可以用来治疗1型和部分2型糖尿病,但由于供体细胞来源匮乏,寻找新的细胞来源成了当务之急。近年来,随着细胞移植和组织工程的日益发展,干细胞研究为新型胰岛的来源开辟了新的途径。本文综述了近年来干细胞分化为胰岛样细胞的研究进展、存在问题和可能的解决途径。     

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20–30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.     

Mechanical stiffness as an improved single-cell indicator of osteoblastic human mesenchymal stem cell differentiation

Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering.     

Immune modulation by mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be derived from various adult tissues with multipotent and self‐renewal abilities. The characteristics of presenting no major ethical concerns, having low immunogenicity and possessing immune modulation functions make MSCs promising candidates for stem cell therapies. MSCs could promote inflammation when the immune system is underactivated and restrain inflammation when the immune system is overactivated to avoid self‐overattack. These cells express many immune suppressors to switch them from a pro‐inflammatory phenotype to an anti‐inflammatory phenotype, resulting in immune effector cell suppression and immune suppressor cell activation. We would discuss the mechanisms governing the immune modulation function of these cells in this review, especially the immune‐suppressive effects of MSCs.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

Prospect of mesenchymal stem cells in therapy of osteoporosis: A review

Osteoporosis is a systemic skeletal disease associated with reduced bone strong point that results in raised fracture risk, with decreased bone strength, leading to reduced bone mineral density and poor bone quality. It is the most common in older females but some men are also at high risk. Although considered as a predictable result of aging, it is can be avoidable and treatable. The existing treatment of osteoporosis mainly contains antiresorptive and anabolic agents. In spite of these improvements, concerns around unusual side-effects of antiresorptive drugs, and the lack of perfect confirmation in maintenance of their long-standing effectiveness is bring about many patients not receiving these drugs. Over the years, the stem cell-based therapy has attained substantial clinical consideration because of its potential to treat numerous diseases. The stem cell therapy has been recommended as a probable therapeutic approach for patients with osteoporosis. Even though the concept of stem cell-based therapy for osteoporosis has caught substantial attention, no clinical trial has been published on humans. The cell studies based on osteoporosis are primarily focused on osteoclastic activity and bone resorption procedures. Earlier, it was on osteoblastogenesis and in recent times, on the differentiation probable of mesenchymal stem cells. In this review, we have summarized the therapeutic role of stem cell-based strategy in osteoporosis.     

Blue photobiomodulation LED therapy impacts SARS-CoV-2 by limiting its replication in Vero cells

The study of any intervention able to counteract SARS-CoV-2 pandemic is considerably envisaged. It was previously shown, in in vitro models of infections, that the LED blue light is able to decrease the viral load of HSV-1 and ZIKV. In our study, LED photobiomodulation therapy (PBMT) at blue wavelengths (450, 454 and 470 nm) was tested in an in vitro model of SARS-CoV-2 infection, employing three experimental settings: SARS-CoV-2 was irradiated and then transferred to cells; already infected cells were irradiated; cells were irradiated prior to infection. A decrement of the viral load was observed when previously infected cells were irradiated with all three tested wavelengths and relevant effects were registered especially at 48 hours post-infection, possibly suggesting that the blue light could interfere with the intracellular viral replication machinery. Our in vitro findings could represent the starting point for translational applications of PBMT as a supportive approach to fight SARS-CoV-2.     

Plasminogen activator inhibitor-1 deficiency suppresses osteoblastic differentiation of mesenchymal stem cells in mice

Plasminogen activator inhibitor-1 (PAI-1) is known as an inhibitor of fibrinolytic system. Previous studies suggest that PAI-1 is involved in the pathogenesis of osteoporosis induced by ovariectomy, diabetes, and glucocorticoid excess in mice. However, the roles of PAI-1 in early-stage osteogenic differentiation have remained unknown. In the current study, we investigated the roles of PAI-1 in osteoblastic differentiation of mesenchymal stem cells (MSCs) using wild-type (WT) and PAI-1-deficient (PAI-1 KO) mice. PAI-1 mRNA levels were increased with time during osteoblastic differentiation of MSCs or mesenchymal ST-2 cells. However, the increased PAI-1 levels declined at the mineralization phase in the experiment using MC3T3-E1 cells. PAI-1 deficiency significantly blunted the expression of osteogenic gene, such as osterix and alkaline phosphatase enhanced by bone morphogenetic protein (BMP)-2 in bone marrow-derived MSCs (BM-MSCs), adipose-tissue-derived MSCs (AD-MSCs), and bone marrow stromal cells of mice. Moreover, a reduction in endogenous PAI-1 levels by small interfering RNA significantly suppressed the expression of osteogenic gene in ST-2 cells. Plasmin did not affect osteoblastic differentiation of AD-MSCs induced by BMP-2 with or without PAI-1 deficiency. PAI-1 deficiency and a reduction in endogenous PAI-1 levels did not affect the phosphorylations of receptor-specific Smads by BMP-2 and transforming growth factor-β in AD-MSCs and ST-2 cells, respectively. In conclusion, we first showed that PAI-1 is crucial for the differentiation of MSCs into osteoblasts in mice.     

ESET histone methyltransferase regulates osteoblastic differentiation of mesenchymal stem cells during postnatal bone development

To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Insulin-producing cells derived from stem cells: recent progress and future directions

Type 1 diabetes is characterized by the selective destruction of pancreatic beta-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to beta-cell loss caused by apoptotic programs, includes beta-cell dedifferentiation and peripheric insulin resistance. beta-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreatic-derived insulin secretion exerts on the body's glycemia. Restoration of damaged beta-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including beta-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic beta-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical applications of this technology.     

Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy

A considerable amount of retrospective data is available that describes putative mesenchymal stem cells (MSCs). However, there is still very little knowledge available that documents the properties of a MSC in its native environment. Although the precise identity of MSCs remains a challenge, further understanding of their biological properties will be greatly advanced by analyzing the mechanisms that govern their self-renewal and differentiation potential. This review begins with the current state of knowledge on the biology of MSCs, specifically with respect to their existence in the adult organism and postulation of their biological niche. While MSCs are considered suitable candidates for cell-based strategies owing to their intrinsic capacity to self-renew and differentiate, there is currently little information available regarding the molecular mechanisms that govern their stem cell potential. We propose here a model for the regulation of MSC differentiation, and recent findings regarding the regulation of MSC differentiation are discussed. Current research efforts focused on elucidating the mechanisms regulating MSC differentiation should facilitate the design of optimal in vitro culture conditions to enhance their clinical utility cell and gene therapy.     

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.