Endothelial Colony-forming Progenitor Cells Research Articles

Manufacture of endothelial colony‐forming progenitor cells from steady‐state peripheral blood leukapheresis using pooled human platelet lysate

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44×10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730×103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.

Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co-Transplantation.

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony-forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long-term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)-mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ-induced HLA-DR expression on ECFCs and MSCs, but both cell types had significantly less HLA-DR in cocultures. ECFC-induced T cell proliferation was abolished after MSC coculture as a result of HLA-DR downregulation and indolamin-2,3-dioxygenase activation. Additionally, allospecific CD8+ T cell-mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA-DR expression following PBMC reconstitution. Crosstalk between UC-derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233-1245.

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Objectives Vitamin D insufficiency is associated with a 5-fold increased risk for development of preeclampsia. We hypothesize that vitamin D plays an important role in the health of endothelial progenitor cells that participate in maternal and placental vasculogenesis and vascular endothelial repair. We asked if vitamin D antagonizes the negative effects of preeclampsia, or a preeclampsia-like environment, on fetal endothelial progenitor cell function. Methods Umbilical cord blood was obtained from preeclamptic and uncomplicated pregnancies. The mononuclear cells were isolated and seeded onto culture plates for outgrowth of endothelial colony forming progenitor cells (ECFCs). Placental explants from uncomplicated term pregnancies were incubated for 48 h under 2%, 8% or 21% O2. Explant conditioned media (CM) was collected and pooled according to oxygen level. Migration, proliferation, and tubule formation of ECFCs was determined in the presence/absence of CM or maternal serum, and 1,25(OH)2 vitamin D3. We inhibited the VEGF signaling pathway with Su5416 and blocked the VDR with VDR antagonist pyridoxal-5-phosphate or antiVDR siRNA. Results The number of cord blood-derived ECFC colonies was decreased in PE compared with controls (P = 0.04). Preeclampsia ECFCs showed reduced proliferation (P Conclusions Physiological concentrations of vitamin D3 promote the migration and capillary-like tubule formation of ECFCs in culture, minimizing negative effects of preeclampsia or preeclampsia-related factors. Such positive effects of vitamin D on progenitor cell function might improve placental and endothelial function and pregnancy outcome. Disclosures L. Brodowski: None. J. Burlakov: None. C.A. Hubel: None. F. von Versen-Hoynck: None.

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.

A Novel Role for Mesenchymal Stem/Progenitor Cells As Hypoxia Sensors During Initiation of Neo-Vasculogenesis in Vivo

Abstract 613 Background:Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods:Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo. To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results:Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O2, while MSPCs stabilized HIF-1α already at 5% O2. In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo. Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions:MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Collagen Receptor-Mediated Mechanochemical Signaling Contributes to Human Pro-Angiogenic Mesenchymal Stem/Progenitor Cell-Induced Neo-Vasculogenesis

Abstract 5196 Background and rationaleHuman mesenchymal stem/progenitor cells (MSPCs) are important tools for tissue repair and regenerative approaches. Co-application of autologous pairs of human MSPCs and endothelial colony forming progenitor cells (ECFCs) drives vessel formation in a mouse model. However, a more detailed mechanistic insight into the contribution of MSPCs to vasculogenesis is required prior to a potential application in clinical trials. The formation of perfused blood vessels following the co-injection of MSPC/ECFC pairs requires directed migration of these cell types through the extracellular matrix. For this, cells must interpret and respond to both biochemical cues and physical parameters of the matrix. Here we aimed at identifying changes in early signaling molecules that could potentially mediate these complex behaviors in ECFCs and MSPCs. ResultsBy antibody array analysis of biopsies from vasculogenic regions we identified the levels of the discoidin domain receptor 2 (DDR2) to be upregulated about twofold in MSPCs in an MSPC/ECFC mixture compared to MSPC-only implants. Expression of DDR2 was confirmed by flow cytometry analysis. Employing immunofluorescence microscopy we showed components of the mechanotransduction and cytoskeleton machinery (Paxillin, ILK, Src, h1CaP, and cortactin) to be abundantly expressed and correctly localized in MSPCs. MSPCs also responded to manipulation of cytoskeletal integrity with phorbol dibutyrate or Y-27632, demonstrating the presence of a tissue transmigration machinery in MSPCs. Applying various three dimensional cell culture strategies we identified significant alterations in MSPC morphology, as well as in the mRNA levels for DDR1 and DDR2, and for miRNAs 29b, 199a, 331 in response to different matrix conditions. ConclusionsOur data suggest a mechanosensitive regulation of MSPC function and we thus conclude that direct or indirect (miRNA-mediated) regulation of the collagen receptors DDR1/2 could have a role in modulating MSPC function during stem-cell induced neo-vascularization in vivo. Disclosures:No relevant conflicts of interest to declare.

Oxygen Sensing Mesenchymal Progenitors Promote Neo-Vasculogenesis in a Humanized Mouse Model In Vivo

Despite insights into the molecular pathways regulating hypoxia-induced gene expression, it is not known which cell types accomplish oxygen sensing during neo-vasculogenesis. We have developed a humanized mouse model of endothelial and mesenchymal progenitor co-transplantation to delineate the cellular compartments responsible for hypoxia response during vasculogenesis. Mesenchymal stem/progenitor cells (MSPCs) accumulated nuclear hypoxia-inducible transcription factor (HIF)-1α earlier and more sensitively than endothelial colony forming progenitor cells (ECFCs) in vitro and in vivo. Hypoxic ECFCs showed reduced function in vitro and underwent apoptosis within 24h in vivo when used without MSPCs. Surprisingly, only in MSPCs did pharmacologic or genetic inhibition of HIF-1α abrogate neo-vasculogenesis. HIF deletion in ECFCs caused no effect. ECFCs could be rescued from hypoxia-induced apoptosis by HIF-competent MSPCs resulting in the formation of patent perfused human vessels. Several angiogenic factors need to act in concert to partially substitute mesenchymal HIF-deficiency. Results demonstrate that ECFCs require HIF-competent vessel wall progenitors to initiate vasculogenesis in vivo and to bypass hypoxia-induced apoptosis. We describe a novel mechanistic role of MSPCs as oxygen sensors promoting vasculogenesis thus underscoring their importance for the development of advanced cellular therapies.

Neo-Vasculogenesis In Vivo Is Facilitated by Oxygen Sensing Mesenchymal Stem and Pogenitor Cells

Abstract 699 Rationale:Vascular repair after hypoxic tissue damage requires a stringent interaction between somatic endothelial colony-forming progenitor cells (ECFCs) and mesenchymal stem and progenitor cells (MSPCs). Stem cell therapy to re-vascularize ischemic tissue has been a promising tool for various therapeutic targets including stroke, myocardial infarction and peripheral artery disease. Despite promising experimental data, therapeutic approaches employing endothelial progenitor cells have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. Hypoxia in ischemic tissue is an extensively studied key factor that influences pro- and anti-angiogenic treatment by driving the revascularization machinery. We and others have shown that despite hypoxic stimulation, ECFCs in vivo only form patent vessels in the presence of MSPCs. Here we show that MSPCs but not ECFCs are the oxygen sensors enabling vasculogenesis in vivo. Methods:Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. In in vitro studies progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen conditions (5% O2) and standard culture conditions (20% O2). ECFC and MSPC interaction in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelete lysate). To investigate the respective roles of MSPCs and ECFCs during vasculogenesis under hypoxia in vivo chemical and genetic inhibitors against protein synthesis (cycloheximide) and HIF-1α (YC-1, shRNA) were employed. Immune histochemistry, immune fluorescence and TUNEL assays were performed on plugs in the time course after transplantation. Results:In vitro studies showed that compared to 20% O2, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced at 5% O2, and even more at 1% O2. Standard culture conditions resulted in a shift in the progenitor hierarchy with an augmented number of high proliferative potential (HPP)-ECFC colonies (60±18% of total colonies) as compared to venous oxygen conditions (9±6%) and a complete loss of HPP-ECFC colonies under severe hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair function in scratch assays and the ability to form vascular-like networks in matrigel assays in vitro were diminished with declining oxygen supply. The re-oxygenation to 20% O2 of ECFCs which where precultured at 1% or 5% O2 led to enhanced proliferation, colony size and function. Single cell analysis revealed that ECFCs stabilized hypoxia-inducing factor-1α (HIF-1α) only at 1% O2 while MSPCs stabilize HIF-1α at 1% O2 as well as 5% O2 conditions. In a mouse model, subcutaneously injected ECFCs underwent apoptosis after 24h and attracted mouse leucocytes. In contrast, ECFCs co-implanted in vivo with MSPCs were rescued from apoptotic death and formed perfused human vessels 7 days after transplantation independent of matrix. Perivascular cells, but not ECFCs, were positive for HIF-1α in vivo. Inhibition of MSPCs but not ECFCs protein synthesis and HIF-1α prior to co-implantation blocked vessel formation. Conclusion:These data demonstrates that hypoxic ECFCs alone show reduced functuionality in vitro and form patent vessels in vivo. In contrast, MSPCs react to the low oxygen environment more sensitively than ECFCs and promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis. Surprisingly, this study shows that therapeutic vasculogenesis can occur independent of endothelial HIF stabilization and protein synthesis. This data indicate that in addition to their established role regulating hematopoiesis, MSPCs oxygen sensing is crucial during vascular regeneration. This suggests a shift of focus from endothelial cells to perivascular cells as a therapeutic target in regenerative medicine and anti-angiogenic therapy. Disclosures:No relevant conflicts of interest to declare.

Endothelial Colony-forming Progenitor Cells: Identification, Isolation, Expansion and Potential Use for Regenerative Cell Therapy

Regenerative medicine is an emerging therapeutic approach which is based on the clinical application of adult stem cells for the repair of organs after toxic, metabolic or ischemic organ injury. Upcoming therapeutic options in regenerative medicine will bring profound challenges for the field of transfusion medicine with regards to new technologies in cell or tissue manipulation and multidisciplinary clinical efforts. In cooperation with hematologists, the teams at trans­fusion medicine departments have been concerned with the identification, isolation and storage of hematopoietic progenitor cells for therapeutic application since more than 20 years. A crucial factor for the successful regeneration of organs after damage due to non-neoplastic reasons is the reconstitution of the functional vascular situation providing sufficient blood perfusion of the target tissues. For this reason clinical research has increasingly focused at circulating endothelial progenitor cells since 15 years. This short article introduces recent findings on “endo­thelial colony-forming progenitor cells (ECFC)” highlighting the role of central actors in post­natal neo-vasculogenesis.

Oxygen Sensing of Mesenchymal Stem and Progenitor Cells Facilitates Neo-Vasculogenesis In Vivo

AbstractAbstract 4313 Background:Vascular homeostasis and regeneration are maintained by proliferating vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs). Despite promising experimental data, regenerative stem cell therapy approaches employing ECFCs have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. We and others have recently shown that ECFC function in vivo requires a stringent interaction with mesenchymal stem and progenitor cells (MSPCs) * [Blood 2009; 113 (26):6716-25]. Co-transplantation of ECFCs and MSPCs is considered to be an advantageous strategy for vascular regenerative medicine.Hypoxia in ischemic tissue is considered to be a key factor influencing pro- and anti-angiogenic treatment by driving the revascularization machinery. In vivo most cells exist under an O2 pressure considerably below air oxygen. In vitro cells are usually expanded under air oxygen and suddenly encounter reduced O2 conditions when re-injected for therapy. Preliminary data suggests that low oxygen conditions differentially regulate stem cell function. We hypothesized that MSPCs act as hypoxia sensors and drive ECFCs to form functioning vessels in vivo. Methods:Adult human ECFCs were isolated and propagated directly from whole venous blood using a novel recovery strategy **[J Vis Exp. 2009;(32) pii: 1524]. MSPCs were isolated from human bone marrow aspirates. During cell culture, pooled human platelet lysate (pHPL) entirely replaced fetal bovine serum. Throughout this study we designated the oxygen level present in vivo in the venous environment as euoxia (41.5±3.4 mmHg). Oxygen levels below euoxia are defined as hypoxia (27.4±7.3 mmHg). Air-oxygen commonly used in standard laboratory practice is above euoxia and is therefore referred to as hyperoxia (139.8±2.9 mmHg). Progenitor cell phenotype, hierarchy, long-term proliferation, wound repair as well as migratory and vasculogenic functions were monitored under euoxia as compared to hypoxic or hyperoxic conditions. Molecular regulation of cellular responses to different oxygen levels was assessed by flow cytometry, immune cytochemistry and proteomic profiling. ECFC and MSPC interactions in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after sub-cutaneous co-implantation in matrigel plugs. Immune histochemistry and TUNEL assays were performed on plugs at day 1, 7 and 14 after transplantation. Results:Compared to hyperoxic standard laboratory conditions in vitro, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced under euoxia, and even more under hypoxic conditions. Hyperoxic conditioning resulted in a shift in progenitor hierarchy with an augmented number of ECFC high proliferative potential (HPP) colonies (60±18% of total colonies) as compared to euoxia (9±6%) and a complete loss of HPP colonies under hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair in scratch assays and matrigel vascular-like network formation in vitro were improved with escalating oxygen supply. The reoxygenation of hypoxic and euoxic ECFCs led to enhanced proliferation and function. Furthermore, MSPCs stabilized hypoxia inducible factor-1α (HIF-1α) under hypoxic as well as euoxic conditions, whereas ECFCs only stabilized HIF-1α when confronted with hypoxia in vitro. In a mouse model, subcutaneously injected ECFCs in matrigel underwent apoptosis after 1 day and attracted mouse leucocytes which infiltrated the matrigel plug. Co-implantation of ECFCs and MSPCs in these matrigel plugs resulted in reduced apoptosis and formation of perfused human vessels as soon as 7 days after transplantation. In this in vivo setting, perivascular cells but not endothelial cells were positive for HIF-1α in immune histochemistry. Background:These data indicate that oxygen levels differentially regulate ECFC and MSPC function during vascular homeostasis and regeneration. While hypoxic ECFCs alone are not able to function in vitro and form patent vessels in vivo, MSPCs react to the low oxygen environment and support ECFCs to perform vessel formation in vivo at least in part by rescuing ECFCs from hypoxia-induced apoptosis. This suggests that oxygen appears to be a key factor in stem cell transplantation and regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion >1x10(8) ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. (3,4) This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.