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Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells.

ABSTRACT: A hierarchy of endothelial colony-forming cells (ECFC) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. ECFC has recently become an attractive target for new vascular regenerative therapies; however, in vitro expansion of ECFC typically depends on the presence of fetal bovine serum (FBS) or fetal calf serum (FCS) in the culture medium, which is not appropriate for its therapeutic application.To identify optimal conditions for in vitro expansion of ECFC, the effects of human endothelial serum-free medium (SFM) supplemented with six pro-angiogenic cytokines and human umbilical cord blood plasma (HCP) were investigated. The in vitro morphology, proliferation, surface antigen expression and in vivo vessel-forming ability were utilized for examining the effects of medium on ECFC.This novel formulation of endothelial cell culture medium allows us, for the first time, to isolate and expand human ECFC efficiently in vitro with a low concentration of HCP (1.5%) and without bovine serum additives. In this serum-reduced medium (SRM), human ECFC colony yields remained quantitatively similar to those cultured in a high concentration (10%) of bovine serum-supplemented medium. SRM-cultured ECFC displayed a robust clonal proliferative ability in vitro and human vessel-forming capacity in vivo.The present study provides a novel method for the expansion of human ECFC in vitro and will help to advance approaches for using the cells in human therapeutic trials.

SUBMITTER: Huang L

PROVIDER: S-EPMC3387926 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells.

Huang Lan L Critser Paul J PJ Grimes Brenda R BR Yoder Mervin C MC

Cytotherapy 20110120 6

<h4>Background aims</h4>A hierarchy of endothelial colony-forming cells (ECFC) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. ECFC has recently become an attractive target for new vascular regenerative therapies; however, in vitro expansion of ECFC typically depends on the presence of fetal bovine serum (FBS) or fetal calf serum (FCS) in the culture medium, which is not appropriate for its therapeutic application.<h4>Methods</h4 ...[more]

PMID: 21250867

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Project description:Angiogenesis is a complicated process in which perivascular cells play important roles. Multipotent mesenchymal stem/stromal cells (MSCs) from distinct tissues have been proved to be proangiogenic and share functional properties and gene expression profiles with perivascular cells. However, different tissues derived MSCs may exhibit different potential for clinical applications. Accordingly, comparative studies on different MSCs are essential. Here, we characterized MSCs from adipose (ADSCs), umbilical cord (UCMSCs), and endometrium (EMSCs) in terms of the surface antigen expression, differentiation ability, and the ability of angiogenesis promotion on endothelial colony-forming cells (ECFCs) both in vitro and in vivo. No significant differences in immunophenotype and differentiation were observed. In addition, three types of MSCs all located around tubular-like structures formed by ECFCs in coculture system on matrigel. But ECFCs seeded on ADSCs monolayer formed more organized capillary-like network than that on UCMSCs or EMSCs. When suspended with ECFCs in matrigel and implanted into nude mice, ADSCs promoted more functional vessel formation after 7 days. Moreover, in murine hindlimb ischemia model, cotransplantation of ECFCs with ADSCs was significantly superior to UCMSCs and EMSCs in promoting perfusion recovery and limb salvage. Furthermore, ADSC-conditioned medium (CM) contained more proangiogenic factors (such as vascular endothelial growth factor-A, platelet-derived growth factor BB, and basic fibroblast growth factor) and less inhibitory factor (such as thrombospondin-1), when compared with UCMSC-CM and EMSC-CM. And ADSC-CM more durably stabilized the vascular-like structures formed by ECFCs on matrigel and promoted ECFCs migration more efficiently. In summary, MSCs from adipose show significantly efficient promotion on angiogenesis both in vitro and in vivo than UCMSCs and EMSCs. Hence, ADSCs may be recommended as a more suitable source for treating hindlimb ischemia.

Project description:BackgroundSuccessful pregnancies are nowadays possible after kidney transplantation but are associated with a higher incidence of maternal and fetal complications. Immunosuppressive therapy causes cardiovascular side effects but must be maintained during pregnancy. Little is known about the consequences of maternal kidney transplantation on offspring's endothelial health. Endothelial colony forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells and are crucial for vascular homeostasis, repair and neovascularization. Therefore, we investigated whether maternal kidney transplantation affects fetal ECFCs' characteristics.MethodsECFCs were isolated from umbilical cord blood of uncomplicated and post-kidney-transplant pregnancies and analyzed for their functional abilities with proliferation, cell migration, centrosome orientation and angiogenesis assays. Further, ECFCs from uncomplicated pregnancies were exposed to either umbilical cord serum from uncomplicated or post-kidney-transplant pregnancies.ResultsPost-kidney-transplant ECFCs showed significantly less proliferation, less migration and less angiogenesis compared to control ECFCs. The presence of post-kidney-transplant umbilical cord serum led to similar functional aberrations of ECFCs from uncomplicated pregnancies.ConclusionsThese pilot data demonstrate differences in ECFCs' biological characteristics in offspring of women after kidney transplantation. Further studies are needed to monitor offspring's long-term cardiovascular development and to assess possible causal relationships with immunosuppressants, uremia and maternal cardiovascular alterations.ImpactPregnancy after kidney transplantation has become more common in the past years but is associated with higher complications for mother and offspring. Little is known of the impact of maternal kidney transplantation and the mandatory immunosuppressive therapy on offspring vascular development. In this study we are the first to address and detect an impairment of endothelial progenitor cell function in offspring of kidney-transplanted mothers. Serum from post-transplant pregnancies also causes negative effects on ECFCs' function. Clinical studies should focus on long-term monitoring of offspring's cardiovascular health.

Project description:BackgroundMany patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology.ResultsDecellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful.ConclusionUsing our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.

Project description:BACKGROUND:Fetal growth restriction (FGR) is associated with an increased susceptibility for various noncommunicable diseases in adulthood, including cardiovascular and renal disease. During FGR, reduced uteroplacental blood flow, oxygen and nutrient supply to the fetus are hypothesized to detrimentally influence cardiovascular and renal programming. This study examined whether developmental programming profiles, especially related to the cardiovascular and renal system, differ in human umbilical vein endothelial cells (HUVECs) collected from pregnancies complicated by placental insufficiency-induced FGR compared to normal growth pregnancies. Our approach, involving transcriptomic profiling by RNA-sequencing and gene set enrichment analysis focused on cardiovascular and renal gene sets and targeted DNA methylation assays, contributes to the identification of targets underlying long-term cardiovascular and renal diseases. RESULTS:Gene set enrichment analysis showed several downregulated gene sets, most of them involved in immune or inflammatory pathways or cell cycle pathways. seven of the 22 significantly upregulated gene sets related to kidney development and four gene sets involved with cardiovascular health and function were downregulated in FGR (n?=?11) versus control (n?=?8). Transcriptomic profiling by RNA-sequencing revealed downregulated expression of LGALS1, FPR3 and NRM and upregulation of lincRNA RP5-855F14.1 in FGR compared to controls. DNA methylation was similar for LGALS1 between study groups, but relative hypomethylation of FPR3 and hypermethylation of NRM were present in FGR, especially in male offspring. Absolute differences in methylation were, however, small. CONCLUSION:This study showed upregulation of gene sets related to renal development in HUVECs collected from pregnancies complicated by FGR compared to control donors. The differentially expressed gene sets related to cardiovascular function and health might be in line with the downregulated expression of NRM and upregulated expression of lincRNA RP5-855F14.1 in FGR samples; NRM is involved in cardiac remodeling, and lincRNAs are correlated with cardiovascular diseases. Future studies should elucidate whether the downregulated LGALS1 and FPR3 expressions in FGR are angiogenesis-modulating regulators leading to placental insufficiency-induced FGR or whether the expression of these genes can be used as a biomarker for increased cardiovascular risk. Altered DNA methylation might partly underlie FPR3 and NRM differential gene expression differences in a sex-dependent manner.

Dataset Information

Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells.

Publications

Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells.

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