Lineage Specification Of Mesenchymal Stem Cells Research Articles

Effects of Different Surface Functionalizations of Silica Nanoparticles on Mesenchymal Stem Cells.

Physicochemical properties of nanoparticles, such as particle size, surface charge, and particle shape, have a significant impact on cell activities. However, the effects of surface functionalization of nanoparticles with small chemical groups on stem cell behavior and function remain understudied. Herein, we incorporated different chemical functional groups (amino, DETA, hydroxyl, phosphate, and sulfonate with charges of +9.5, + 21.7, -14.1, -25.6, and -37.7, respectively) to the surface of inorganic silica nanoparticles. To trace their effects on mesenchymal stem cells (MSCs) of rat bone marrow, these functionalized silica nanoparticles were used to encapsulate Rhodamine B fluorophore dye. We found that surface functionalization with positively charged and short-chain chemical groups facilitates cell internalization and retention of nanoparticles in MSCs. The endocytic pathway differed among functionalized nanoparticles when tested with ion-channel inhibitors. Negatively charged nanoparticles mainly use lysosomal exocytosis to exit cells, while positively charged nanoparticles can undergo endosomal escape to avoid scavenging. The cytotoxic profiles of these functionalized silica nanoparticles are still within acceptable limits and tolerable. They exerted subtle effects on the actin cytoskeleton and migration ability. Last, phosphate-functionalized nanoparticles upregulate osteogenesis-related genes and induce osteoblast-like morphology, implying that it can direct MSCs lineage specification for bone tissue engineering. Our study provides insights into the rational design of biomaterials for effective drug delivery and regenerative medicine.

Adipocyte hypertrophy in mesenchymal stem cells from infants of mothers with obesity.

Fat content of adipocytes derived from infant umbilical cord mesenchymal stem cells (MSCs) predicts adiposity in children through 4 to 6 years of age. This study tested the hypothesis that MSCs from infants born to mothers with obesity (Ob-MSCs) exhibit adipocyte hypertrophy and perturbations in genes regulating adipogenesis compared with MSCs from infants of mothers with normal weight (NW-MSCs). Adipogenesis was induced in MSCs embedded in three-dimensional hydrogel structures, and cell size and number were measured by three-dimensional imaging. Proliferationand protein markers of proliferation and adipogenesis in undifferentiated and adipocyte differentiating cells were measured. RNA sequencing was performed to determine pathways linked to adipogenesis phenotype. In undifferentiated MSCs, greater zinc finger protein (Zfp)423 protein content was observed in Ob- versus NW-MSCs. Adipocytes from Ob-MSCs were larger but fewer than adipocytes from NW-MSCs. RNA sequencing analysis showed that Zfp423 protein correlated with mRNA expression of genes enriched for cell cycle, MSC lineage specification, inflammation, and metabolism pathways. MSC proliferation was not different before differentiation but declined faster in Ob-MSCs upon adipogenic induction. Ob-MSCs have an intrinsic propensity for adipocyte hypertrophy and reduced hyperplasia during adipogenesis in vitro, perhaps linked to greater Zfp423 content and changes in cell cycle pathway gene expression.

Inflammatory Cytokine TNF-a Controls Mesenchymal Stem Fate by Regulating JMJD3 Expression

Introduction: Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate in to osteoblast, adipocytes and chondrocytes. Lineage specification of MSC is governed by various systemic hormones, systemic and local growth factors and cytokines. TNF-α is an inflammatory cytokine produced at the site of tissue injuries and known to regulates MSC migration and differentiation. However, its role on lineage specification and differentiation of MSCs remain complex and elusive. In this study we explored the same utilizing human bone marrow and adipocyte derived MSCs. Experimental Methods: Human MSCs derived from bone marrow and adipocytes were differentiated in to osteoblast and adipocytes in the presence or absence of TNF-α. Expressions of osteoblast and adipocyte differentiation markers were assessed by qRT-PCR. The key epigenetic factor of lineage specification JMJD3 was depleted in MSCs utilizing lentiviral ShRNA. Results: TNF-α promoted the osteoblastic and inhibited the adipogenic differentiation of MSC as assessed by Alizarin and oil red O staining, respectively. Consistently, while inducing the key osteogenic factors, TNF- α repressed the adipogenic markers in MSCs. Mechanistically, TNF-α regulates MSC fate by inducing lysine-specific demethylase JMJD3/KDM6B, which is a key epigenetic factor that determines mesenchymal stem cell lineage specification. ShRNA mediated knockdown of JMD3 in MSCs inhibited TNF- α mediated activation and inhibition of osteogenic and adipogenic differentiation, respectively. Conclusion: Our study uncovers the novel mechanisms of TNF-α mediated MSC lineage commitment and differentiation and thus highlight JMJD3 as mediator of TNF-α actions in MSCs.

Mesenchymal stem cell and bone marrow mononuclear cell therapy for cardiomyopathy: From bench to bedside.

To date, stem cell-based therapies for cardiac diseases have not achieved any significant clinical accomplishment. Globally, numerous patients are currently treated with autologous stem cells. The safety and practicality of this technique have been well-examined, its disadvantages have been recognized, and many trials have been proposed. Inadequate description of the implemented cell types, a variety of cell-handling proficiencies, and concerning factors related to autologous stem cells have been known as the central elements restricting the approval of cell-based therapies. The idea that bone marrow (BM)-derived cells could be applied to regenerate and cure damage in various organs is the basis for bone marrow mononuclear cell (BMMNC) therapy for heart disease. Mesenchymal stem cells (MSCs) are a part of the BMMNCs; on one hand, they have the capability to differentiate into various tissues, and, on the other, their immunomodulatory effects have been considered and clinically confirmed in different experiments. In this review, we summarize the knowledge obtained by trials in which mesenchymal cell-based therapy has been practiced. Furthermore, we accentuate the developments in the purification and lineage specification of MSCs as well as BMMNCs that have influenced the progress of future stem cell-based therapies with special attention on cardiovascular disease.

Mechanisms of stem cell based cardiac repair-gap junctional signaling promotes the cardiac lineage specification of mesenchymal stem cells

Different subtypes of bone marrow-derived stem cells are characterized by varying functionality and activity after transplantation into the infarcted heart. Improvement of stem cell therapeutics requires deep knowledge about the mechanisms that mediate the benefits of stem cell treatment. Here, we demonstrated that co-transplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) led to enhanced synergistic effects on cardiac remodeling. While HSCs were associated with blood vessel formation, MSCs were found to possess transdifferentiation capacity. This cardiomyogenic plasticity of MSCs was strongly promoted by a gap junction-dependent crosstalk between myocytes and stem cells. The inhibition of cell-cell coupling significantly reduced the expression of the cardiac specific transcription factors NKX2.5 and GATA4. Interestingly, we observed that small non-coding RNAs are exchanged between MSCs and cardiomyocytes in a GJ-dependent manner that might contribute to the transdifferentiation process of MSCs within a cardiac environment. Our results suggest that the predominant mechanism of HSCs contribution to cardiac regeneration is based on their ability to regulate angiogenesis. In contrast, transplanted MSCs have the capability for intercellular communication with surrounding cardiomyocytes, which triggers the intrinsic program of cardiogenic lineage specification of MSCs by providing cardiomyocyte-derived cues.

Retinoid X Receptor Activation Alters the Chromatin Landscape To Commit Mesenchymal Stem Cells to the Adipose Lineage.

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

Long non-coding RNA HoxA-AS3 interacts with EZH2 to regulate lineage commitment of mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and are involving in diverse cellular processes. However, their roles in reprogramming of gene expression profiles during lineage commitment and maturation of mesenchymal stem cells (MSCs) remain poorly understood. In the current study, we characterize the expression of a lncRNA, HoxA-AS3, during the differentiation of MSCs. We showed that HoxA-AS3 is increased upon adipogenic induction of MSCs, while HoxA-AS3 remains unaltered during osteogenic induction. Silencing of HoxA-AS3 in MSCs resulted in decreased adipogenesis and expression of adipogenic markers, PPARG, CEBPA, FABP4 and ADIPOQ. Conversely, knockdown of HoxA-AS3 expression in MSCs exhibited an enhanced osteogenesis and osteogenic markers expression, including RUNX2, SP7, COL1A1, IBSP, BGLAP and SPP1. Mechanistically, HoxA-AS3 interacts with Enhancer Of Zeste 2 (EZH2) and is required for H3 lysine-27 trimethylation (H3K27me3) of key osteogenic transcription factor Runx2. Our data reveal that HoxA-AS3 acts as an epigenetic switch that determines the lineage specification of MSC.

The Lineage Specification of Mesenchymal Stem Cells Is Directed by the Rate of Fluid Shear Stress.

The effective regulation of fluid shear stress (FSS) on the lineage specification of mesenchymal stem cells (MSCs) remains to be addressed. We hypothesized that when MSCs are recruited to musculoskeletal system following stimulation, their differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation-selective channels (MSCCs), intracellular calcium levels, and F-actin. To this end, MSCs were exposed to laminar FSS linearly increased from 0 to 10 dyn/cm(2) in 0, 2, or 20 min and maintained at 10 dyn/cm(2) for a total of 20 min (termed as ΔSS 0-0', 0-2', and 0-20', respectively, representing more physiological (0-0') and non-physiological (0-2' and 0-20') ΔSS treatments). Our results showed 0-0' facilitated MSC differentiation towards chondrogenic and not osteogenic phenotype, by promoting moderate intracellular calcium concentration ([Ca(2+) ]i ) increase from the calcium channels with the exception of MSCCs or intracellular calcium stores, and F-actin organization. In contrast, 0-2' promoted MSCs towards osteogenic and not chondrogenic phenotype, by inducing significant [Ca(2+) ]i increase mainly from the MSCCs, and F-actin assembly. However, 0-20' elicited the modest osteogenic and chondrogenic phenotypes, as it induced the lowest [Ca(2+) ]i increase mainly from MSCCs, and F-actin assembly. Our results suggest that compared to the more physiological ΔSS, the non-physiological ΔSS favors [Ca(2+) ]i influx from MSCCs. An appropriate non-physiological ΔSS (0-2') even elicits a large [Ca(2+) ]i influx from the MSCCs that reverses the lineage specification of MSCs, providing validation for the high mechanosensitivity of MSCs and guidance for training osteoporosis and osteoarthritis patients. J. Cell. Physiol. 231: 1752-1760, 2016. © 2015 Wiley Periodicals, Inc.

Histone methyltransferases and demethylases: regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3–9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

Detachment of Mesenchymal Stem Cells with Trypsin/EDTA Has No Effect on Apoptosis Detection

Hui and colleagues have raised the concern that the detection of cell surface Annexin V staining on mesenchymal stem cells (MSC) following treatment with the enhancer of Zeste homology 2 (EZH2) inhibitor, DZNep (Sequoia Research, Pangbourne, United States, http:// www.seqchem.com/) may have been compromised due to the use of Trypsin/EDTA to detach the adherent cells in our recent publication “EZH2 and KDM6A Act as an Epigenetic Switch to Regulate MSC lineage specification” [1]. Specifically, it is proposed that EDTA could affect the structure of the plasma membrane and chelate Ca preventing the binding of Annexin V protein to the phosphatidylserine [2–4]. However, different manufacturers specify the use of Trypsin/EDTA, and not EDTA-free trypsin for detaching adherent cells prior to staining for Annexin V, where Trypsin/EDTA has been used by various studies for a wide range of adherent cell types [5–12]. While the Letter to the Editor by Hui and colleagues, published in this article, use the example that DZNep has been previously shown to induced apoptosis in human colon cancer stem cells [13], our study [1] found that DZNep had no effect on the apoptosis status of human bone marrow-derived MSC, in accord with other studies reporting that DZNeP is not harmful for nontransformed cells at tumor-inhibiting doses [14]. The discrepancy in DZNep sensitivity between normal stem cells and cancer stem cells may be due to aberrant levels of EZH2 in cancer cells required for survival [15, 16]. In order to determine whether EDTA influences Annexin V binding on MSC, we performed the Annexin V protocol with either Trypsin/EDTA (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) as described in our published study [1], or with EDTA-free Collagenase I (2 mg/ml Worthington Biochemical Corporation, NJ, United States of America, http://www.worthington-biochem. com/default.html). Two different human MSC cultures were treated with 5 mM of the potent apoptotic drug, doxorubicin (Sapphire Biosciences, NSW, Australia, http://www.sapphirebiosci ence.com/) for 24 hours prior to Annexin V staining. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then washed with Hanks Balanced Salt Solution (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) supplemented with 5% fetal calf serum (FCS; SAFC Biosciences, Sydney, NSW, Australia, http://www.safcglobal.com/safc-global/en-us/ home.html). The cells were further washed twice with cold phosphate buffered saline prior to cell staining for Annexin V as specified in the manufacturer’s protocol (Annexin V conjugates Alexa Fluro 488, for apoptosis detection, Life Technologies, VIC, Australia, http:// www.lifetechnologies.com/au/). Flow cytometry was used to determine the levels of Annexin V staining under each condition. Cells treated with doxorubicin without Annexin V Figure 1. Comparable expression of Annexin V on doxorubicin treated MSC detached by Trypsin/EDTA or Collagenase I. Two different human bone marrow-derived MSC cultures were treated with 5 mM doxorubicin for 24 hours. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then stained for Annexin V. Assessment of Annexin V levels was performed by flow cytometric analysis. Data represent mean fluorescence intensity from three replicate experiments. Abbreviation: MSC, mesenchymal stem cells. Mesechymal Stem Cell Laboratory, School of Health Sciences, Faculty of Health Sciences and Centre for Stem Cell Research, University of Adelaide, South Australia, Australia

Cytoskeletal and Focal Adhesion Influences on Mesenchymal Stem Cell Shape, Mechanical Properties, and Differentiation Down Osteogenic, Adipogenic, and Chondrogenic Pathways

Mesenchymal stem cells (MSCs) hold great potential for regenerative medicine and tissue-engineering applications. They have multipotent differentiation capabilities and have been shown to differentiate down various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and possibly neurons. The majority of approaches to control the MSC fate have been via the use of chemical factors in the form of growth factors within the culture medium. More recently, it has been understood that mechanical forces play a significant role in regulating MSC fate. We and others have shown that mechanical stimuli can control MSC lineage specification. The cytoskeleton is known to play a large role in mechanotransduction, and a growing number of studies are showing that it can also contribute to MSC differentiation. This review analyzes the significant contribution of actin and integrin distribution, and the smaller role of microtubules, in regulating MSC fate. Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions. Different mechanical stimuli can be implemented to alter these cytoskeletal patterns and encourage MSC differentiation to the desired lineage.