Pericyte-Like Progenitors Show High Immaturity and Engraftment Potential as Compared with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) and pericyte progenitors (PPs) are both perivascular cells with similar multipotential properties regardless of tissue of origin. We compared the phenotype and function of the 2 cell types derived from the same bone-marrow samples but expanded in their respective media – pericyte conditions (endothelial cell growth medium 2 [EGM-2]) for PPs and standard medium (mesenchymal stem cell medium [MSM]) for MSCs. After 3 weeks of culture, whatever the expansion medium, all cells showed similar characteristics (MSC markers and adipo-osteo-chondroblastic differentiation potential), although neuronal potential was greater in EGM-2– than MSM-cultured cells. As compared with MSM-cultured MSCs, EGM-2–cultured PPs showed higher expression of the pericyte-specific antigen 3G5 than a-smooth muscle actin. In addition, EGM-2–cultured PPs showed an immature phenotype, with upregulation of stemness OCT4 and SOX2 proteins and downregulation of markers of osteoblastic, chondroblastic, adipocytic and vascular smooth muscle lineages. Despite having less effective in vitro immunosuppression capacities than standard MSCs, EGM-2–cultured PPs had higher engraftment potentials when combined with biomaterials heterotopically-transplanted in Nude mice. Furthermore, these engrafted cells generated more collagen matrix and were preferentially perivascular or lined trabeculae as compared with MSM-cultured MSCs. In conclusion, EGM-2–cultured PPs are highly immature cells with increased plasticity and engraftment potential

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1.

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5

<i>In vivo</i> detection of cells of human origin.

<p>Alu sequences were checked in cells from microsections of BCP transplants seeded with PPs cultured with EGM-2 (<b>A–C</b>) or MSCs with MSM (<b>D</b>). (<b>A</b>) Human cells were depicted by red staining in nuclei (arrowheads). (<b>B</b>) and (<b>C</b>) enlargement of the squared area in (<b>A</b>). Human cells lined BCP trabeculae, formed fibrous tissue, or were at the abluminal position of vessels (<b>B, C</b>). (<b>D</b>) Very few cells of human origin were detected with seeding of MSM-cultured MSCs. (<b>E</b>) Quantification of human vs host cells involved counting the number of Alu+ nuclei (*p<0.001) in more than 10 fields per transplant and 3 transplants.</p

Expression of stemness markers.

<p>Quantitative RT-PCR (<b>A</b>) and western blot (<b>B</b>) analysis of the mRNA and protein expression, respectively, of stemness specific markers OCT4A, SOX2 and quantitative RT-PCR analysis of NANOG mRNA expression. Data are mean±SD from 3 experiments. *p<0.01; **p<0.001 compared with PPs. Protein extracts of cells from 3 different BM cultures (BM1, BM2, BM3) of PPs vs MSCs were tested. β-actin was a positive internal control. ND: not determined. MEL1: protein extract of human embryonic stem cell line MEL1 as positive control.</p

Expression of mesenchymal lineage markers.

<p>(<b>A</b>) Quantitative RT-PCR of relative mRNA expression of markers of chondrogenesis (<i>COL2a1</i>, <i>SOX9</i>, <i>SOX6</i>, <i>DLX2</i>), adipogenesis (<i>PPARγ2</i>, <i>ADIPOQ</i>, <i>LEP</i>), osteogenesis (<i>ALPL</i>, <i>PTHR1</i>, <i>RUNX2</i>, <i>DLX5</i>, <i>OSC</i>, <i>OPG</i>, <i>RANKL</i>), and vascular smooth muscle (VSM) (<i>EMX2</i>, <i>CNN3</i>, <i>ELN</i>, <i>MYOC</i>). Expression was relative to that of <i>GAPDH</i> and depicted as fold change relative to that for pericyte progenitors (PPs). Data are mean±SEM from 6 experiments. *p<0.01; **p<0.001 compared with PPs. (<b>B</b>) Western blot analysis of protein level of RUNX2 in 3 different BM productions (BM1, BM2, and BM3) of bone-marrow MSCs and PPs. β-actin was a positive internal control.</p

<i>In vivo</i> investigation of stem/progenitor cell seeding in mice.

<p>EGM-2–derived PPs or MSM-derived MSCs were seeded into biphasic calcium phosphate (BCP) biomaterial discs and inserted in the back of mice (<b>A</b>). Several BCP discs were not loaded with cells as negative controls (<b>B</b>). After 8 weeks, all discs were removed and evaluated for collagen matrix deposition within transplants and human cell engraftment. Vessels and bone collagen were observed after hematoxylin (left) or Masson’s trichrome (right) staining, respectively. Dotted black lines indicate the outer border of discs. Red arrows show vessels, and blue arrows show bone matrix. (<b>C</b>) BCP discs loaded with EGM-2–cultured PPs show trabeculae lined with cells engulfed within matrix on Masson’s trichrome staining (green fibers).</p