A chemically defined biomimetic surface for enhanced isolation efficiency of high-quality human mesenchymal stromal cells under xenogeneic/serum-free conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by (i) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell expansion; (ii) donor-specific characteristics, including MSC frequency/quality, that decline with disease state and increasing age; and (iii) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which poses safety and consistency issues. METHODS: To overcome these limitations while enabling robust MSC production with constant high yield and quality, the authors developed a chemically defined biomimetic surface coating called isoMATRIX (denovoMATRIX GmbH, Dresden, Germany) and tested its performance during isolation of MSCs. RESULTS: The isoMATRIX facilitates the isolation of significantly higher numbers of MSCs in xenogeneic (xeno)/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. The high proliferation rates can be maintained up to 5 passages after isolation and cells even benefit from a switch towards a proliferation-specific MSC matrix (myMATRIX MSC) (denovoMATRIX GmbH, Dresden, Germany). CONCLUSION: In sum, isoMATRIX promotes enhanced xeno/serum-free and chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Class I and IIa histone deacetylases have opposite effects on sclerostin gene regulation.

Adult bone mass is controlled by the bone formation repressor sclerostin (SOST). Previously, we have shown that intermittent parathyroid hormone (PTH) bone anabolic therapy involves SOST expression reduction by inhibiting myocyte enhancer factor 2 (MEF2), which activates a distant bone enhancer. Here, we extended our SOST gene regulation studies by analyzing a role of class I and IIa histone deacetylases (HDACs), which are known regulators of MEF2s. Expression analysis using quantitative PCR (qPCR) showed high expression of HDACs 1 and 2, lower amounts of HDACs 3, 5, and 7, low amounts of HDAC4, and no expression of HDACs 8 and 9 in constitutively SOST-expressing UMR106 osteocytic cells. PTH-induced Sost suppression was associated with specific rapid nuclear accumulation of HDAC5 and co-localization with MEF2s in nuclear speckles requiring serine residues 259 and 498, whose phosphorylations control nucleocytoplasmic shuttling. Increasing nuclear levels of HDAC5 in UMR106 by blocking nuclear export with leptomycin B (LepB) or overexpression in transient transfection assays inhibited endogenous Sost transcription and reporter gene expression, respectively. This repressor effect of HDAC5 did not require catalytic activity using specific HDAC inhibitors. In contrast, inhibition of class I HDAC activities and expression using RNA interference suppressed constitutive Sost expression in UMR106 cells. An unbiased comprehensive search for involved HDAC targets using an acetylome analysis revealed several non-histone proteins as candidates. These findings suggest that PTH-mediated Sost repression involves nuclear accumulation of HDAC inhibiting the MEF2-dependent Sost bone enhancer, and class I HDACs are required for constitutive Sost expression in osteocytes.

Mef2c deletion in osteocytes results in increased bone mass.

Myocyte enhancer factors 2 (MEF2) are required for expression of the osteocyte bone formation inhibitor Sost in vitro, implying these transcription factors in bone biology. Here, we analyzed the in vivo function of Mef2c in osteocytes in male and female mice during skeletal growth and aging. Dmp1-Cre-induced Mef2c deficiency led to progressive decreases in Sost expression by 40% and 70% in femoral cortical bone at 3.5 months and 5 to 6 months of age. From 2 to 3 months onward, bone mass was increased in the appendicular and axial skeleton of Mef2c mutant relative to control mice. Cortical thickness and long bone and vertebral trabecular density were elevated. To assess whether the increased bone mass was related to the decreased Sost expression, we characterized 4-month-old heterozygous Sost-deficient mice. Sost heterozygotes displayed similar increases in long bone mass and density as Mef2c mutants, but the relative increases in axial skeletal parameters were mostly smaller. At the cellular level, bone formation parameters were normal in 3.5-month-old Mef2c mutant mice, whereas bone resorption parameters were significantly decreased. Correspondingly, cortical expression of the anti-osteoclastogenic factor and Wnt/ß-catenin target gene osteoprotegerin (OPG) was increased by 70% in Mef2c mutant males. Furthermore, cortical expression of the Wnt signaling modulators Sfrp2 and Sfrp3 was strongly deregulated in both sexes. In contrast, heterozygous Sost deficient males displayed mildly increased osteoblastic mineral apposition rate, but osteoclast surface and cortical expression of osteoclastogenic regulators including OPG were normal and Sfrp2 and Sfrp3 were not significantly changed. Together, our data demonstrate that Mef2c regulates cortical Sfrp2 and Sfrp3 expression and is required to maintain normal Sost expression in vivo. Yet, the increased bone mass phenotype of Mef2c mutants is not directly related to the reduced Sost expression. We identified a novel function for Mef2c in control of adult bone mass by regulation of osteoclastic bone resorption.

Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm.

FGFRL1 is a recently discovered member of the fibroblast growth factor receptor family that is lacking the intracellular tyrosine kinase domain. To elucidate the function of the novel receptor, we created mice with a targeted disruption of the Fgfrl1 gene. These mice develop normally until term, but die within a few minutes after birth due to respiratory failure. The respiratory problems are explained by a significant reduction in the size of the diaphragm muscle, which is not sufficient to inflate the lungs after birth. The remaining portion of the diaphragm muscle appears to be well developed and innervated. It consists of differentiated myofibers with nuclei at the periphery. Fast and slow muscle fibers occur in normal proportions. The myogenic regulatory factors MyoD, Myf5, myogenin and Mrf4 and the myocyte enhancer factors Mef2A, Mef2B, Mef2C and Mef2D are expressed at normal levels. Experiments with a cell culture model involving C2C12 myoblasts show that Fgfrl1 is expressed during the late stages of myotube formation. Other skeletal muscles do not appear to be affected in the Fgfrl1 deficient mice. Thus, Fgfrl1 plays a critical role in the development of the diaphragm.

Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor.

FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.