Progression of human bone marrow stromal cells into both osteogenic and adipogenic lineages is differentially regulated by structural conformation of collagen I matrix via distinct signaling pathways

Adult human bone marrow stromal cells (BMSCs) containing or consisting of mesenchymal stem cells (MSCs) are an important source in tissue homeostasis and repair. Although many processes involved in their differentiation into diverse lineages have been deciphered, substantial inroads remain to be gained to synthesize a complete regulatory picture. The present study suggests that structural conformation of extracellular collagen I, the major organic matrix component in musculoskeletal tissues, plays, along with differentiation stimuli, a decisive role in the selection of differentiation lineage. It introduces a novel concept which proposes that structural transition of collagen I matrix regulates cell differentiation through distinct signaling pathways specific for the structural state of the matrix. Thus, on native collagen I matrix inefficient adipogenesis is p38-independent, whereas on its denatured counterpart, an efficient adipogenesis is primarily regulated by p38 kinase. Inversely, osteogenic differentiation occurs efficiently on native, but not on denatured collagen I matrix, with a low commencement threshold on the former and a substantially higher one on the latter. Osteogenesis on collagen I matrices in both structural conformations is fully dependent on ERK. However, whereas on native collagen I matrix osteogenic differentiation is Hsp90-dependent, on denatured collagen I matrix it is Hsp90-independent. The matrix conformation-mediated regulation appears to be one of the mechanisms determining differentiation lineage of BMSCs. It allows a novel interpretation of the bone remodeling cycle, explains the marked physiological aging-related adipogenic shift in musculoskeletal tissues, and can be a principal contributor to adipogenic shift seen in a number of clinical disorders.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Molecular mechanisms of natural killer cell activation in response to cellular stress

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.     

Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell

A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.     

Characterization of cell signaling,morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.     

Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage

Human mesenchymal stem cell (hMSC) research has grown exponentially in the last decade. The ability to process and preserve these cells is vital to their use in stem cell therapy. As such, understanding the complex, molecular-based stress responses associated with biopreservation is necessary to improve outcomes and maintain the unique stem cell properties specific to hMSC. In this study hMSC were exposed to cold storage (4 °C) for varying intervals in three different media. The addition of resveratrol or salubrinal was studied to determine if either could improve cell tolerance to cold. A rapid elevation in apoptosis at 1 h post-storage as well as increased levels of necrosis through the 24 h of recovery was noted in samples. The addition of resveratrol resulted in significant improvements to hMSC survival while the addition of salubrinal revealed a differential response based on the media utilized. Decreases in both apoptosis and necrosis together with decreased cell stress/death signaling protein levels were observed following modulation. Further, ER stress and subsequent unfolded protein response (UPR) stress pathway activation was implicated in response to hMSC hypothermic storage. This study is an important first step in understanding hMSC stress responses to cold exposure and demonstrates the impact of targeted molecular modulation of specific stress pathways on cold tolerance thereby yielding improved outcomes. Continued research is necessary to further elucidate the molecular mechanisms involved in hypothermic-induced hMSC cell death. This study has demonstrated the potential for improving hMSC processing and storage through targeting select cell stress pathways.     

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.     

3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart.     

The cellular form of the prion protein is involved in controlling cell cycle dynamics,self‐renewal,and the fate of human embryonic stem cell differentiation

Prion protein (PrP^C), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrP^C in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline‐regulated lentiviral vectors that up‐regulate or suppresses PrP^C expression. Here, we show that expression of PrP^C in pluripotent hESCs cultured under self‐renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrP^C in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over‐expression of PrP^C in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrP^C is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self‐renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrP^C is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self‐renewal state, control cell proliferation activity, and define stem cell fate.     

Matrix remodeling as stem cell recruitment event: A novel in vitro model for homing of human bone marrow stromal cells to the site of injury shows crucial role of extracellular collagen matrix

The goal of the present study was to devise an in vitro model suitable for investigations of the homing of mesenchymal stem cells to sites of injury. Such a model was designed on the basis of a “transwell” assay, with an insert seeded with human bone marrow stromal cells and a well with a desired cell type. To mimic physiological environment and to simulate “injury”, cells in a well were maintained not only on tissue culture plastic but also on collagens I and IV, major matrix components in musculoskeletal and adipose tissues respectively, and subjected to a severe thermal stress. The results obtained showed a massive translocation of bone marrow stromal cells through the inserts' membrane toward the “injury” site. Unexpectedly, it emerged that collagen matrix is essential in producing such a migration. The results obtained suggest that upon injury cells secrete a substance which interacts with collagen matrix to produce a homing agent. The substance in question appears to be a protease and its interaction with the collagen matrix appears to be a digestion of the latter into fragments shown to be chemotactic. Both AEBSF, an inhibitor of serine proteases, and leupeptin, an inhibitor of cysteine proteases as well as of trypsin-like serine proteases, but not the broad spectrum MMP inhibitor marimastat, significantly inhibit the observed homing effect and this inhibition is not due to cytotoxicity. Moreover, immunoprecipitation of HTRA1, a trypsin-like serine protease known to be secreted by cells differentiating into all three major mesenchymal lineages and by stressed cells in general and shown to degrade a number of matrix proteins including collagen, significantly diminished the homing effect. The data suggest that this protease is a major contributor to the observed chemotaxis of bone marrow stromal cells. The present study indicates that collagen fragments can mediate the migration of bone marrow stromal cells. The results also suggest that, at least in musculoskeletal and in adipose tissues, matrix remodeling occurrences, usually closely associated with tissue remodeling, should also be regarded as potential stem cells recruitment events.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

The hypoxic peri-arteriolar glioma stem cell niche,an integrated concept of five types of niches in human glioblastoma

Glioblastoma is the most lethal primary brain tumor and poor survival of glioblastoma patients is attributed to the presence of glioma stem cells (GSCs). These therapy-resistant, quiescent and pluripotent cells reside in GSC niches, which are specific microenvironments that protect GSCs against radiotherapy and chemotherapy. We previously showed the existence of hypoxic peri-arteriolar GSC niches in glioblastoma tumor samples. However, other studies have described peri-vascular niches, peri-hypoxic niches, peri-immune niches and extracellular matrix niches of GSCs. The aim of this review was to critically evaluate the literature on these five different types of GSC niches. In the present review, we describe that the five niche types are not distinct from one another, but should be considered to be parts of one integral GSC niche model, the hypoxic peri-arteriolar GSC niche. Moreover, hypoxic peri-arteriolar GSC niches are structural and functional look-alikes of hematopoietic stem cell (HSC) niches in the bone marrow. GSCs are maintained in peri-arteriolar niches by the same receptor-ligand interactions as HSCs in bone marrow. Our concept should be rigidly tested in the near future and applied to develop therapies to expel and keep GSCs out of their protective niches to render them more vulnerable to standard therapies.