A. V. Makarov

Umbilical Cord as Prospective Source for Mesenchymal Stem Cell-Based Therapy

The paper presents current evidence on the properties of human umbilical cord-derived mesenchymal stem cells, including origin, proliferative potential, plasticity, stability of karyotype and phenotype, transcriptome, secretome, and immunomodulatory activity. A review of preclinical studies and clinical trials using this cell type is performed. Prospects for the use of mesenchymal stem cells, derived from the umbilical cord, in cell transplantation are associated with the need for specialized biobanking and transplant standardization criteria.

Role of VEGF-A in angiogenesis promoted by umbilical cord-derived mesenchymal stromal/stem cells: in vitro study

BackgroundMesenchymal stromal/stem cells derived from human umbilical cord (UC-MSCs) uniquely combine properties of embryonic and postnatal MSCs and may be the most acceptable, safe, and effective source for allogeneic cell therapy e.g. for therapeutic angiogenesis. In this report we describe pro-angiogenic properties of UC-MSCs as manifested in vitro.MethodsUC-MSCs were isolated from human Wharton’s jelly by enzymatic digestion. Presence of soluble forms of VEGF-A in UC-MSC-conditioned media was measured by ELISA. Effects of the conditioned media on human umbilical vein-derived endothelial EA.hy926 cells proliferation were measured by MTT-assay; changes in cell motility and directed migration were assessed by scratch wound healing and transwell chamber migration assays. Angiogenesis was modeled in vitro as tube formation on basement membrane matrix. Progressive differentiation of MSCs to endothelioid progeny was assessed by CD31 immunostaining.ResultsAlthough no detectable quantities of soluble VEGF-A were produced by UC-MSCs, the culture medium, conditioned by the UC-MSCs, effectively stimulated proliferation, motility, and directed migration of EA.hy926 cells. In 2D culture, UC-MSCs were able to acquire CD31+ endothelial cell-like phenotype when stimulated by EA.hy926-conditioned media supplemented with VEGF-A165. UC-MSCs were capable of forming unstable 2D tubular networks either by themselves or in combinations with EA.hy926 cells. Active spontaneous sprouting from cell clusters, resulting from disassembling of such networks, was observed only in the mixed cultures, not in pure UC-MSC cultures. In 3D mode of sprouting experimentation, structural support of newly formed capillary-like structures was provided by UC-MSCs that acquired the CD31+ phenotype in the absence of exogenous VEGF-A.ConclusionThese data suggest that a VEGF-A-independent paracrine mechanism and at least partially VEGF-A-independent differentiation mechanism are involved in the pro-angiogenic activity of UC-MSCs.

Bone marrow-derived multipotent stromal cells promote myocardial fibrosis and reverse remodeling of the left ventricle.

Cell therapy is increasingly recognized as a beneficial practice in various cardiac conditions, but its fundamentals remain largely unclear. The fates of transplanted multipotent stromal cells in postinfarction cardiac microenvironments are particularly understudied. To address this issue, labeled multipotent stromal cells were infused into rat myocardium at day 30 after myocardial infarction, against the background of postinfarction cardiosclerosis. Therapeutic effects of the transplantation were assessed by an exercise tolerance test. Histological examination at 14 or 30 days after the transplantation was conducted by means of immunostaining and quantitative image analysis. An improvement in the functional status of the cardiovascular system was observed after both the autologous and the allogeneic transplantations. Location of the label-positive cells within the heart was restricted to the affected part of myocardium. The transplanted cells could give rise to fibroblasts or myofibroblasts but not to cardiac myocytes or blood vessel cells. Both types of transplantation positively influenced scarring processes, and no expansion of fibrosis to border myocardium was observed. Left ventricular wall thickening associated with reduced dilatation index was promoted by transplantation of the autologous cells. According to the results, multipotent stromal cell transplantation prevents adverse remodeling and stimulates left ventricular reverse remodeling.

Molecular Survey of Cell Source Usage during Subtotal Hepatectomy-Induced Liver Regeneration in Rats.

Proliferation of hepatocytes is known to be the main process in the hepatectomy-induced liver regrowth; however, in cases of extensive loss it may be insufficient for complete recovery unless supported by some additional sources e.g. mobilization of undifferentiated progenitors. The study was conducted on rat model of 80% subtotal hepatectomy; the objective was to evaluate contributions of hepatocytes and resident progenitor cells to the hepatic tissue recovery via monitoring specific mRNA and/or protein expression levels for a panel of genes implicated in growth, cell differentiation, angiogenesis, and inflammation. Some of the genes showed distinctive temporal expression patterns, which were loosely associated with two waves of hepatocyte proliferation observed at 2 and 7 days after the surgery. Focusing on genes implicated in regulation of the progenitor cell activity, we came across slight increases in expression levels for Sox9 and two genes encoding tumor necrosis factor-like cytokine TWEAK (Tnfsf12) and its receptor Fn14 (Tnfrsf12a). At the same time, no increase in numbers of cytokeratin 19-positive (CK19+) cells was observed in periportal areas, and no CK19+ cells were found in hepatic plates. Since CK19 is thought to be a specific marker of both cholangiocytes and the hepatic progenitor cells, the data indicate a lack of activation of the resident progenitor cells during recovery of hepatic tissue after 80% subtotal hepatectomy. Thus, proliferation of hepatocytes invariably makes the major contribution to the hepatic tissue recovery, although in the cases of subtotal loss this contribution is distinctively modulated. In particular, induction of Sox9 and TWEAK/Fn14 regulatory pathways, conventionally attributed to progenitor cell activation, may incidentally stimulate mitotic activity of hepatocytes.

Effect of Endothelial Cells on Angiogenic Properties of Multipotent Stromal Cells from the Umbilical Cord during Angiogenesis Modeling in the Basement Membrane Matrix

Short-term cell culturing on basement membrane matrix is a common and very convenient in vitro model of angiogenesis. We studied the possibility of interaction of multipotent stromal cells from the umbilical cord and Ea.hy926 endothelial cells on this model at the early and late periods of the experiment. Multipotent stromal cells alone and in combination with endothelial cells formed an unstable tubular network. Clusters formed after its disassembling later became the sprouting centers in co-culture of the two cell types, but not in pure culture of multipotent stromal cells. Multipotent stromal cells with CD31+ phenotype constitute the structural basis of newly formed stable 3D capillary-like network. Prolongation of the time of culturing and combination of the two in vitro models of angiogenesis (tubulogenesis and sprouting) allowed more complete assessment of the angiogenic potential of multipotent stromal cells.

Expression of Cytokine Genes and Growth Factors in Rat Lungs and Kidneys after Subtotal Hepatectomy.

Expression of il1b, il6, il10, tnfa, hgf, tgfb, vegf, and fgf2 genes in the lungs and kidneys was examined on rat model of liver regeneration after subtotal hepatectomy. Enhanced expression of il6, il10, tnfa, hgf, and fgf2 genes was detected at the early terms after 80% liver resection.

Effect of Multipotent Stromal Cells on the Function of Cell Mitochondria in Regenerating Liver

Intrasplenic allogeneic transplantation of multipotent stromal cells from the umbilical cord stimulates hepatocyte proliferation and promotes recovery of liver weight in rats after subtotal resection (80% organ weight). It can be hypothesized that this effect of multipotent stromal cells is due to more rapid recovery of the number of mitochondria and normalization of mitochondrial function of liver hepatocytes.

Dynamics of macrophage populations of the liver after subtotal hepatectomy in rats

BackgroundIn many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the ‘small for size’ liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats.ResultsThe data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages – the resident Kupffer cells with CD68+CX1CR3−CD11b− phenotype.ConclusionsThe delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the ‘proregeneratory’ M2 macrophage-specific marker CD206 in the course of regeneration.

Role of Progenitor Cells in Liver Regeneration after Subtotal Resection

In the liver of rats subjected to subtotal liver resection (80% organ weight), the expression of sox9 gene and SOX9 protein content increased and cells with hepatocyte morphology expressing SOX9 appeared; the proportion of cells expressing cytokeratin-19 also increased. Based on these data, we cannot completely exclude the involvement of resident progenitor cells and hepatocyte reprogramming in liver regeneration after subtotal resection, however, the contribution of these processes seems to be insignificant. The leading mechanism of liver mass recovery after subtotal resection is proliferation of hepatocytes.

Multipotent stromal cells stimulate liver regeneration by influencing the macrophage polarization in rat

AIM To investigate the influence of the umbilical cord-derived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome. METHODS The MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with РКН26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT. RESULTS Intrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study. CONCLUSION We found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked.