Bithiah Grace Jaganathan

Rho Inhibition Induces Migration of Mesenchymal Stromal Cells

Although mesenchymal stromal cells (MSCs) are being increasingly used as cell therapeutics in clinical trials, the mechanisms that regulate their chemotactic migration behavior are incompletely understood. We aimed to better define the ability of the GTPase regulator of cytoskeletal activation, Rho, to modulate migration induction in MSCs in a transwell chemotaxis assay. We found that culture‐expanded MSCs migrate poorly toward exogenous phospholipids lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) in transwell assays. Moreover, plasma‐induced chemotactic migration of MSCs was even inhibited after pretreatment with LPA. LPA treatment activated intracellular Rho and increased actin stress fibers in resident MSCs. Very similar cytoskeletal changes were observed after microinjection of a cDNA encoding constitutively active RhoA (RhoAV14) in MSCs. In contrast, microinjection of cDNA encoding Rho inhibitor C3 transferase led to resolution of actin stress fibers, appearance of a looser actin meshwork, and increased numbers of cytoplasmic extensions in the MSCs. Surprisingly, in LPA‐pretreated MSCs migrating toward plasma, simultaneous addition of Rho inhibitor C2I‐C3 reversed LPA‐induced migration suppression and led to improved migration. Moreover, addition of Rho inhibitor C2I‐C3 resulted in an approximately 3‐ to 10‐fold enhancement of chemotactic migration toward LPA, S1P, as well as platelet‐derived growth factor or hepatocyte growth factor. Thus, inhibition of Rho induces rearrangement of actin cytoskeleton in MSCs and renders them susceptible to induction of migration by physiological stimuli.

Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway

BackgroundMesenchymal Stem Cells (MSC) are important candidates for therapeutic applications due to their ex vivo proliferation and differentiation capacity. MSC differentiation is controlled by both intrinsic and extrinsic factors and actin cytoskeleton plays a major role in the event. In the current study, we tried to understand the initial molecular mechanisms and pathways that regulate the differentiation of MSC into osteocytes or adipocytes.ResultsWe observed that actin modification was important during differentiation and differentially regulated during adipogenesis and osteogenesis. Initial disruption of actin polymerization reduced further differentiation of MSC into osteocytes and osteogenic differentiation was accompanied by increase in ERK1/2 and p38 MAPK phosphorylation. However, only p38 MAPK phosphorylation was down regulated upon inhibition of actin polymerization which as accompanied by decreased CD49E expression.ConclusionTaken together, our results show that actin modification is a pre-requisite for MSC differentiation into osteocytes and adipocytes and osteogenic differentiation is regulated through p38 MAPK phosphorylation. Thus by modifying their cytoskeleton the differentiation potential of MSC could be controlled which might have important implications for tissue repair and regeneration.

Collagen Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.

Effects of MSC co-injection on the reconstitution of aplastic anemia patient following hematopoietic stem cell transplantation

Effects of MSC co-injection on the reconstitution of aplastic anemia patient following hematopoietic stem cell transplantation

Multiple roles of CD90 in cancer

THY1 (CD90) is a 25–37-kDa heavily N-glycosylated, glycophosphatidylinositol (GPI) anchored cell surface protein. It is usually expressed on thymocytes, mesenchymal stem cells, hematopoietic stem cells, natural killer cells, neurons, endothelial cells, renal glomerular mesangial cells, follicular dendritic cells, fibroblasts, and myofibroblasts. It has been found to regulate cell adhesion, migration, apoptosis, axon growth, cell-cell and cell-matrix interactions, T-cell activation, and fibrosis. Several reports have shown that CD90 has an important role in cancer in regulating cancer cell proliferation, metastasis, and angiogenesis. There are also evidences that CD90 is an important prognostic marker in many cancers. Consequently, therapies that target CD90 have great promise in treating many cancers. However, several studies also indicate a contradictory role for CD90, where it acts as a tumor suppressor. In this review, we summarize the expression, function of CD90 in different cancers and its possible use as a biomarker or a therapeutic target in cancer. The challenges and future prospects for the use of CD90 for clinical applications are also discussed in this review.

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

BackgroundHematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.ResultsBy utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.ConclusionThe current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.

Human mesenchymal stromal cells senesce with exogenous OCT4

BACKGROUND AIMS The identification of mesenchymal stromal cells (MSC) from bone marrow by Friedenstein et al. dates back to 1970, but many questions remain unanswered about the biology of these cells. MSC have a wide clinical application because of their differentiation capacity and immunosuppressive properties. They are capable of self-renewal; however, the mechanism is poorly understood. The embryonic transcription factor Octamer binding transcription factor 4 (Oct4) has been suggested as an indicator of multipotency for mouse MSC. OCT4 has also been studied extensively for its involvement in self-renewal of embryonic stem cells and in cancer cells. METHODS Using a lentiviral-inducible vector, we modulated OCT4 expression in human MSC and studied its effect on the biology of these cells. RESULTS Surprisingly, we found that the overexpression of OCT4 induced early senescence, as evidenced by increased expression of P14 and P16(INK4A) in MSC. CONCLUSIONS These results indicate a differential role for exogenously expressed human OCT4 in adult and embryonic systems.

Adhesion to stromal cells mediates imatinib resistance in chronic myeloid leukemia through ERK and BMP signaling pathways

Chronic myeloid leukemia (CML) is characterized by abnormal proliferation of myeloid cells which when untreated leads to bone marrow failure. Imatinib mesylate (IM) is the first line of therapy for treatment of CML and results in remission in most cases. However, a significant percentage of patients develop chemoresistance to IM, which might be due to the presence of chemoresistant cells in the bone marrow. In the current study, we explored the role of cell-cell interaction of CML cells with the bone marrow stromal cells in the development of chemoresistance in CML. We found that the stromal cells offered long-term chemoprotection to the CML cells from the apoptotic effect of IM. These stroma interacting CML cells were maintained in a non-proliferative stage and had increased ERK1/2 and SMAD1/8 phosphorylation levels. Prolonged interaction of CML cells with the stromal cells in the presence of IM resulted in the acquisition of stroma-free chemoresistance to IM treatment. However, inhibition of actin cytoskeleton, ERK1/2 and SMAD signaling abrogated the chemoresistance acquisition and sensitized the chemoresistant CML cells to IM induced apoptosis.

K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

Bone marrow (BM) microenvironment plays an important role in normal and malignant hematopoiesis. As a consequence of interaction with the leukemic cells, the stromal cells of the bone marrow become deregulated in their normal function and gene expression. In our study, we found that mesenchymal stem cells (MSC) from BM of chronic myeloid leukemia (CML) patients have defective osteogenic differentiation and on interaction with K562 CML cells, the normal MSC showed reduced osteogenic differentiation. On interaction with K562 cells or its secreted factors, MSC acquired phenotypic abnormalities and secreted high levels of IL6 through NFκB activation. The MSC derived secreted factors provided a survival advantage to CML cells from imatinib induced apoptosis. Thus, a therapy targeting stromal cells in addition to leukemia cells might be more effective in eliminating CML cells.

Mesenchymal Stem Cells from Human Extra Ocular Muscle Harbor Neuroectodermal Differentiation Potential

Mesenchymal stem cells (MSC) have been proposed as suitable candidates for cell therapy for neurological disorderssince they exhibit good neuronal differentiation capacity. However, for better therapeutic outcomes, it is necessary to isolate MSC from a suitable tissue sourcethat posses high neuronal differentiation. In this context, we isolated MSC from extra ocular muscle (EOM) tissue and tested the in vitro neuronal differentiation potential. In the current study, EOM tissue derived MSC were characterized and compared with bone marrow derived MSC. We found that EOM derived MSC proliferated as a monolayer and showed similarities in morphology, growth properties and cell surface marker expression with bone marrow derived MSC and expressed high levels of NES, OCT4, NANOG and SOX2 in its undifferentiated state. They also expressed embryonic cell surface marker SSEA4 and their intracellular mitochondrial distribution pattern was similar to that of multipotent stem cells. Although EOM derived MSC differentiated readily into adipocytes, osteocytes and chondrocytes, they differentiated more efficiently into neuroectodermal cells. The differentiation into neuroectodermal cellswas confirmed by the expression of neuronal markers NGFR and MAP2B. Thus, EOM derived MSC might be good candidates for stem cell based therapies for treating neurodegenerative diseases.