Menstrual Blood-Derived Stem Cell Paracrine Factors Possess Stimulatory Effects on Chondrogenesis In Vitro and Diminish the Degradation of Articular Cartilage during Osteoarthritis.

Articular cartilage is an avascular tissue with a limited capacity for self-regeneration, leading the tissue to osteoarthritis (OA). Mesenchymal stem cells (MSCs) are promising for cartilage tissue engineering, as they are capable of differentiating into chondrocyte-like cells and secreting a number of active molecules that are important for cartilage extracellular matrix (ECM) synthesis. The aim of this study was to evaluate the potential of easily accessible menstrual blood-derived MSC (MenSC) paracrine factors in stimulating bone marrow MSC (BMMSCs) chondrogenic differentiation and to investigate their role in protecting cartilage from degradation in vitro. MenSCs and BMMSCs chondrogenic differentiation was induced using four different growth factors: TGF-ß3, activin A, BMP-2, and IGF-1. The chondrogenic differentiation of BMMSCs was stimulated in co-cultures with MenSCs and cartilage explants co-cultured with MenSCs for 21 days. The chondrogenic capacity of BMMSCs was analyzed by the secretion of four growth factors and cartilage oligomeric matrix protein, as well as the release and synthesis of cartilage ECM proteins, and chondrogenic gene expression in cartilage explants. Our results suggest that MenSCs stimulate chondrogenic response in BMMSCs by secreting activin A and TGF-ß3 and may have protective effects on cartilage tissue ECM by decreasing the release of GAGs, most likely through the modulation of activin A related molecular pathway. In conclusion, paracrine factors secreted by MenSCs may turn out to be a promising therapeutical approach for cartilage tissue protection and repair.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

The Effect of Heat Shock on Myogenic Differentiation of Human Skeletal-Muscle-Derived Mesenchymal Stem/Stromal Cells.

Muscle injuries, degenerative diseases and other lesions negatively affect functioning of human skeletomuscular system and thus quality of life. Therefore, the investigation of molecular mechanisms, stimulating myogenic differentiation of primary skeletal-muscle-derived mesenchymal stem/stromal cells (SM-MSCs), is actual and needed. The aim of the present study was to investigate the myogenic differentiation of CD56 (neural cell adhesion molecule, NCAM)-positive and -negative SM-MSCs and their response to the non-cytotoxic heat stimulus. The SM-MSCs were isolated from the post operation muscle tissue, sorted by flow cytometer according to the CD56 biomarker and morphology, surface profile, proliferation and myogenic differentiation has been investigated. Data show that CD56(+) cells were smaller in size, better proliferated and had significantly higher levels of CD146 (MCAM) and CD318 (CDCP1) compared with the CD56(-) cells. At control level, CD56(+) cells significantly more expressed myogenic differentiation markers MYOD1 and myogenin (MYOG) and better differentiated to the myogenic direction. The non-cytotoxic heat stimulus significantly stronger stimulated expression of myogenic markers in CD56(+) than in CD56(-) cells that correlated with the multinucleated cell formation. Data show that regenerative properties of CD56(+) SM-MSCs can be stimulated by an extracellular stimulus and be used as a promising skeletal muscle regenerating tool in vivo.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Improves Energetic Status and Cardiomyogenic Differentiation of Human Dilated Myocardium-Derived Primary Mesenchymal Cells.

BACKGROUND: In this study the effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) on the energetic status and cardiomyogenic differentiation of human healthy and dilated myocardium-derived mesenchymal stromal cells (hmMSC) have been investigated. METHODS: The hmMSC were isolated from the healthy and dilated post-operation heart biopsies by explant outgrowth method. Cell proliferation, HDAC activity, mitochondrial membrane potential, and level of adenosine triphosphate (ATP) were evaluated. The effect of SAHA on mitochondrial parameters has been investigated also by Seahorse XF analyzer and cardiomyogenic differentiation was confirmed by the expression of transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T and alpha cardiac actin at gene and protein levels. RESULTS: Dilated myocardium-derived hmMSC had almost 1.5 folds higher HDAC activity compared to the healthy cells and significantly lower mitochondrial membrane potential and ATP level. HDAC class I and II inhibitor SAHA improved energetic status of mitochondria in dilated myocardium-isolated hmMSC and increased expression of cardiac specific proteins during 14 days of exposure of cells to SAHA. CONCLUSIONS: HDAC inhibitor SAHA can be a promising therapeutic for dilated cardiomyopathy (DCM). Dilated hmMSC exposed to SAHA improved energetic status and, subsequently, cardiomyogenic differentiation. Data suggest that human dilated myocardium-derived MSC still have cardio tissue regenerative potential, which might be stimulated by HDAC inhibitors.

Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac Development, Degeneration and Regeneration.

Cardiovascular diseases are the most common cause of human death in the developing world. Extensive evidence indicates that various toxic environmental factors and unhealthy lifestyle choices contribute to the risk, incidence and severity of cardiovascular diseases. Alterations in the genetic level of myocardium affects normal heart development and initiates pathological processes leading to various types of cardiac diseases. Homeobox genes are a large and highly specialized family of closely related genes that direct the formation of body structure, including cardiac development. Homeobox genes encode homeodomain proteins that function as transcription factors with characteristic structures that allow them to bind to DNA, regulate gene expression and subsequently control the proper physiological function of cells, tissues and organs. Mutations in homeobox genes are rare and usually lethal with evident alterations in cardiac function at or soon after the birth. Our understanding of homeobox gene family expression and function has expanded significantly during the recent years. However, the involvement of homeobox genes in the development of human and animal cardiac tissue requires further investigation. The phenotype of human congenital heart defects unveils only some aspects of human heart development. Therefore, mouse models are often used to gain a better understanding of human heart function, pathology and regeneration. In this review, we have focused on the role of homeobox genes in the development and pathology of human heart as potential tools for the future development of targeted regenerative strategies for various heart malfunctions.