Hyaluronic Acid Prevents Fusion of Brain Tumor-Derived Spheroids and Selectively Alters Their Gene Expression Profile.

Hyaluronic acid (HA), a major glycosaminoglycan of the brain extracellular matrix, modulates cell behaviors through binding its receptor, Cd44. In this study, we assessed the influence of HA on high-grade brain tumors in vitro. The model comprised cell cultures derived from six rodent carcinogen-induced brain tumors, forming 3D spheroids prone to spontaneous fusion. Supplementation of the standard culture medium with 0.25% HA significantly inhibited the fusion rates, preserving the shape and size uniformity of spheroids. The 3D cultures were assigned to two groups; a Cd44lo group had a tenfold decreased relative expression of Cd44 than another (Cd44hi) group. In addition, these two groups differed by expression levels of Sox2 transcription factor; the correlation analysis revealed a tight negative association for Cd44 and Sox2. Transcriptomic responses of spheroids to HA exposure also depended on Cd44 expression levels, from subtle in Cd44lo to more pronounced and specific in Cd44hi, involving cell cycle progression, PI3K/AKT/mTOR pathway activation, and multidrug resistance genes. The potential HA-induced increase in brain tumor 3D models' resistance to anticancer drug therapy should be taken into account when designing preclinical studies using HA scaffold-based models. The property of HA to prevent the fusion of brain-derived spheroids can be employed in CNS regenerative medicine and experimental oncology to ensure the production of uniform, controllably fusing neurospheres when creating more accurate in vitro brain models.

Gene-Expression Patterns of Tumor and Peritumor Tissues of Smoking and Non-Smoking HPV-Negative Patients with Head and Neck Squamous Cell Carcinoma.

We studied the gene-expression patterns in specimens of tumor and peritumor tissue biopsies of 26 patients with head and neck carcinomas depending on smoking status. Histological and immunohistochemical examinations verified that all tumors belonged to the "classical" subgroup of head and neck carcinomas, and the HPV-negative tumor status was confirmed. The expression of 28 tumor-associated genes determined by RT-PCR was independent of patients' sex or age, TNM status, degree of differentiation, or tissue localization. Moreover, in peritumor tissue, none of the 28 genes were differentially expressed between the groups of smoking and nonsmoking patients. During oncotransformation in both studied groups, there were similar processes typical for HNSCC progression: the expression levels of paired keratins 4 and 13 were reduced, while the expression levels of keratin 17 and CD44 were significantly increased. However, further investigation revealed some distinctive features: the expression of the genes EGFR and TP63 increased significantly only in the nonsmoking group, and the expression of IL6, CDKN2A, EGF, and PITX1 genes changed only in the smoking group. In addition, correlation analysis identified several clusters within which genes displayed correlations in their expression levels. The largest group included 10 genes: TIMP1, TIMP2, WEE1, YAP, HIF1A, PI3KCA, UTP14A, APIP, PTEN, and SLC26A6. The genetic signatures associated with smoking habits that we have found may serve as a prerequisite for the development of diagnostic panels/tests predicting responses to different therapeutic strategies for HNSCC.

In Vitro Models of Head and Neck Cancer: From Primitive to Most Advanced.

For several decades now, researchers have been trying to answer the demand of clinical oncologists to create an ideal preclinical model of head and neck squamous cell carcinoma (HNSCC) that is accessible, reproducible, and relevant. Over the past years, the development of cellular technologies has naturally allowed us to move from primitive short-lived primary 2D cell cultures to complex patient-derived 3D models that reproduce the cellular composition, architecture, mutational, or viral load of native tumor tissue. Depending on the tasks and capabilities, a scientific laboratory can choose from several types of models: primary cell cultures, immortalized cell lines, spheroids or heterospheroids, tissue engineering models, bioprinted models, organoids, tumor explants, and histocultures. HNSCC in vitro models make it possible to screen agents with potential antitumor activity, study the contribution of the tumor microenvironment to its progression and metastasis, determine the prognostic significance of individual biomarkers (including using genetic engineering methods), study the effect of viral infection on the pathogenesis of the disease, and adjust treatment tactics for a specific patient or groups of patients. Promising experimental results have created a scientific basis for the registration of several clinical studies using HNSCC in vitro models.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model.

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Chain-End Functionalization of Poly(ε-caprolactone) for Chemical Binding with Gelatin: Binary Electrospun Scaffolds with Improved Physico-Mechanical Characteristics and Cell Adhesive Properties.

Composite biocompatible scaffolds, obtained using the electrospinning (ES) technique, are highly promising for biomedical application thanks to their high surface area, porosity, adjustable fiber diameter, and permeability. However, the combination of synthetic biodegradable (such as poly(ε-caprolactone) PCL) and natural (such as gelatin Gt) polymers is complicated by the problem of low compatibility of the components. Previously, this problem was solved by PCL grafting and/or Gt cross-linking after ES molding. In the present study, composite fibrous scaffolds consisting of PCL and Gt were fabricated by the electrospinning (ES) method using non-functionalized PCL1 or NHS-functionalized PCL2 and hexafluoroisopropanol as a solvent. To provide covalent binding between PCL2 and Gt macromolecules, NHS-functionalized methyl glutarate was synthesized and studied in model reactions with components of spinning solution. It was found that selective formation of amide bonds, which provide complete covalent bonding of Gt in PCL/Gt composite, requires the presence of weak acid. With the use of the optimized ES method, fibrous mats with different PCL/Gt ratios were prepared. The sample morphology (SEM), hydrolytic resistance (FT-IR), cell adhesion and viability (MTT assay), cell penetration (fluorescent microscopy), and mechanical characteristics of the samples were studied. PCL2-based films with a Gt content of 20 wt% have demonstrated the best set of properties.

Cryopreservation of Tissue-Engineered Scaffold-Based Constructs: from Concept to Reality.

Creation of scaffold-based tissue-engineered constructs (SB TECs) is costly and requires coordinated qualified efforts. Cryopreservation enables longer shelf-life for SB TECs while enormously enhancing their availability as medical products. Regenerative treatment with cryopreserved SB TECs prepared in advance (possibly prêt-à-porter) can be started straight away on demand. Animal studies and clinical trials indicate similar levels of safety for cryopreserved and freshly prepared SB TECs. Although cryopreservation of such constructs is more difficult than that of cell suspensions or tissues, years of research have proved the principal possibility of using ready-to-transplant SB TECs after prolonged cryostorage. Cryopreservation efficiency depends not only on the sheer viability of adherent cells on scaffolds after thawing, but largely on the retention of proliferative and functional properties by the cells, as well as physical and mechanical properties by the scaffolds. Cryopreservation protocols require careful optimization, as their efficiency depends on multiple parameters including cryosensitivity of cells, chemistry and architecture of scaffolds, conditions of cell culture before freezing, cryoprotectant formulations, etc. In this review we discuss recent achievements in SB TEC cryopreservation as a major boost for the field of tissue engineering and biobanking.

Comparative Analysis of the Transcriptome, Proteome, and miRNA Profile of Kupffer Cells and Monocytes.

Macrophage populations in most mammalian organs consist of cells of different origin. Resident macrophages originate from erythromyeloid precursors of the yolk sac wall; maintenance of the numbers of such macrophages in postnatal ontogenesis is practically independent of bone marrow haematopoiesis. The largest populations of the resident macrophages of embryonic origin are found in the central nervous system (microglia) and liver (Kupffer cells). In contrast, skin dermis and mucous membranes become predominantly colonized by bone marrow-derived monocytes that show pronounced functional and phenotypic plasticity. In the present study, we compared Kupffer cells and monocytes using the immunophenotype, gene expression profile, proteome, and pool of microRNA. The observed differences did not consider the resident liver macrophages as purely M2 macrophages or state that monocytes have purely M1 features. Monocytes show signs of high plasticity and sensitivity to pathogen-associated molecular patterns (e.g., high levels of transcription for Tlr 2, 4, 7, and 8). In contrast, the resident liver macrophages were clearly involved in the regulation of specific organ functions (nitrogen metabolism, complement system protein synthesis).

Molecular mechanisms of splenectomy-induced hepatocyte proliferation.

Functional and anatomical connection between the liver and the spleen is most clearly manifested in various pathological conditions of the liver (cirrhosis, hepatitis). The mechanisms of the interaction between the two organs are still poorly understood, as there have been practically no studies on the influence exerted by the spleen on the normal liver. Mature male Sprague-Dawley rats of 250-260 g body weight, 3 months old, were splenectomized. The highest numbers of Ki67+ hepatocytes in the liver of splenectomized rats were observed at 24 h after the surgery, simultaneously with the highest index of Ki67-positive hepatocytes. After surgical removal of the spleen, expression of certain genes in the liver tissues increased. A number of genes were upregulated in the liver at a single time point of 24 h, including Ccne1, Egf, Tnfa, Il6, Hgf, Met, Tgfb1r2 and Nos2. The expression of Ccnd1, Tgfb1, Tgfb1r1 and Il10 in the liver was upregulated over the course of 3 days after splenectomy. Monitoring of the liver macrophage populations in splenectomized animals revealed a statistically significant increase in the proportion of CD68-positive cells in the liver (as compared with sham-operated controls) detectable at 24 h and 48 h after the surgery. The difference in the liver content of CD68-positive cells between splenectomized and sham-operated animals evened out by day 3 after the surgery. No alterations in the liver content of CD163-positive cells were observed in the experiments. A decrease in the proportion of CD206-positive liver macrophages was observed at 48 h after splenectomy. The splenectomy-induced hepatocyte proliferation is described by us for the first time. Mechanistically, the effect is apparently induced by the removal of spleen as a major source of Tgfb1 (hepatocyte growth inhibitor) and subsequently supported by activation of proliferation factor-encoding genes in the liver.

Regenerative medicine of pancreatic islets.

The pancreas became one of the first objects of regenerative medicine, since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted. The number of people living with diabetes mellitus is currently approaching half a billion, hence the crucial relevance of new methods to stimulate regeneration of the insulin-secreting ß-cells of the islets of Langerhans. Natural restrictions on the islet regeneration are very tight; nevertheless, the islets are capable of physiological regeneration via ß-cell self-replication, direct differentiation of multipotent progenitor cells and spontaneous α- to ß- or δ- to ß-cell conversion (trans-differentiation). The existing preclinical models of ß-cell dysfunction or ablation (induced surgically, chemically or genetically) have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation. The ultimate goal, sufficient level of functional activity of ß-cells or their substitutes can be achieved by two prospective broad strategies: ß-cell replacement and ß-cell regeneration. The "regeneration" strategy aims to maintain a preserved population of ß-cells through in situ exposure to biologically active substances that improve ß-cell survival, replication and insulin secretion, or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-ß- to ß-cell conversion. The "replacement" strategy implies transplantation of ß-cells (as non-disintegrated pancreatic material or isolated donor islets) or ß-like cells obtained ex vivo from progenitors or mature somatic cells (for example, hepatocytes or α-cells) under the action of small-molecule inducers or by genetic modification. We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally active ß-cells, the innermost hope of millions of people globally.

Activated Macrophages of Monocytic Origin Predominantly Express Proinflammatory Cytokine Genes, Whereas Kupffer Cells Predominantly Express Anti-Inflammatory Cytokine Genes.

In the central nervous system and in the liver, the macrophage populations are represented exclusively by descendants of the hematopoietic progenitor cells of the yolk sac. The reasons for such differential distribution of macrophages are not fully understood. We found that, as can be judged by corresponding changes in the expression of CD86 and CD163 markers, the transient macrophages of monocytic lineage are more sensitive to activating stimuli. The two macrophage populations have distinct patterns of gene expression, which is particularly noticeable for M1- and M2-associated genes. For instance, Kupffer cells more readily develop and longer maintain the elevated expression levels of Il4, Il10, and Il13 upon the activation; by contrast, the macrophages of monocytic lineage express Il1b, Il12a, and Tnfα upon the activation. The obtained results allow us to conclude that the in vitro activated Kupffer cells of the liver are committed to M2 phenotype, whereas the in vitro activated monocyte-derived macrophages show a typical M1 behavior. These observations are likely to reflect the situation in the in vivo microenvironments.

Evaluation of resorbable polydioxanone and polyglycolic acid meshes in a rat model of ventral hernia repair.

The objective of this study was to evaluate physical, mechanical, and biological properties of the polydioxanone (PDO) monofilament meshes and polyglycolide (PGA) polyfilament meshes in comparison with Permacol® implants. In rat experimental model, a 1.5 × 2.0 cm defect in abdominal wall was reconstructed by using the Permacol surgical implant or knitted meshes produced from either PDO monofilament, or PGA multifilament. The implant sites were assessed for the tensile strength and the extents of material resorption, host inflammatory response and host tissue replacement on days 3, 10, 30, or 60 after the surgery. The PDO and PGA meshes were rapidly pervaded by the host connective tissue with elements of skeletal muscle histogenesis. The degree of adhesions was significantly higher in the Permacol group. All of the prostheses underwent resorption, which correlated with gradual decreases in the overall tensile strength of the site and the Col1a1 gene expression level. Elevated expression of Fgf2 gene maintained longer in the PDO group, and the Mmp9 gene expression level in this group was higher than in the other groups. Gene expression levels of inflammatory cytokines were higher in the Permacol group. The foreign body giant cell numbers were lower in the PDO and Permacol groups than in the PGA group. Minimal macrophage infiltration with predominance of M2 cells was observed in the PDO group. Overall, the PDO prosthesis turned out to be significantly better than the PGA or Permacol prostheses by a number of indicators of biocompatibility and efficacy. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 652-663, 2019.

Umbilical cord-derived mesenchymal stromal/stem cells enhance recovery of surgically induced skeletal muscle ischemia in a rat model.

This study delves into possible mechanisms underlying the stimulating influence of UC-MSCs transplantation on functional and structural recovery of ischemic skeletal muscles. Limb ischemia was created in Sprague-Dawley rats by excision of femoral and popliteal arteries. Allogeneic rat PKH26-labeled UC-MSCs were administered by direct intramuscular injection. Animals of experimental group responded to the transplantation by improvement in their locomotor function as assessed by the rotarod performance test on day 9 and 29 after transplantation. Histomorphometric analysis showed that relative area of the lesions in the experimental group was significantly smaller than in the control group at all time points during the observation. Calculated densities of microcirculation vessels within the lesions were significantly higher in the experimental group than in the control group on day 10 after transplantation. Only a part of the transplanted allogeneic UC-MSCs survived within the ischemic muscle tissue, and a considerable portion of these surviving cells were found alongside the VEGF-producing preserved muscle fibers. The PKH26 label was not found within the walls of capillaries or larger blood vessels. The administration of allogeneic UC-MSCs significantly increased the proportion of M2 macrophages, exhibiting proangiogenic and anti-inflammatory properties, for at least 10 days following the transplantation.

Umbilical cord tissue cryopreservation: a short review.

In this review we present current evidence on the possibility of umbilical cord tissue cryopreservation for subsequent clinical use. Protocols for obtaining umbilical cord-derived vessels, Wharton's jelly-based grafts, multipotent stromal cells, and other biomedical products from cryopreserved umbilical cords are highlighted, and their prospective clinical applications are discussed. Examination of recent literature indicates we should expect high demand for cryopreservation of umbilical cord tissues in the near future.

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

BACKGROUND: In 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. RESULTS: The 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. CONCLUSIONS: The 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.

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.

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.

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.

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

BACKGROUND: Mesenchymal 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. METHODS: UC-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. RESULTS: Although 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. CONCLUSION: These 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.

Elimination of allogeneic multipotent stromal cells by host macrophages in different models of regeneration.

Allogeneic multipotent stromal cells were previously thought to be poorly recognized by host immune system; the prolonged survival in host environments was explained by their immune privileged status. As long as the concept is currently reconsidered, the routes of elimination of allogeneic multipotent stromal cells by host immunity must be taken into account. This is necessary for correct comprehension of their therapeutic action. The study was focused upon survival of umbilical cord-derived allogeneic multipotent stromal cells in different rat models of tissue regeneration induced by partial hepatectomy or by critical limb ischemia. The observations were carried out by means of vital labeling of the cells with PKH26 prior to injection, in combination with differential immunostaining of host macrophages with anti-CD68 antibody. According to the results, allogeneic multipotent stromal cells are specifically eliminated by host immune system; the efficacy can reach 100%. Massive clearance of transplanted cells by host macrophages is accompanied by appropriation of the label by the latter, and this is a pronounced case of misleading presentation of exogenous label by host cells. The study emphasizes the role of macrophages in host response and also the need of additional criteria for correct data interpretation.