Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+ Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression

Abstract Background: The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. Recently, we have demonstrated that there are 2 distinct types of EPCs from UCB having different biologic properties for angiogenic capabilities in vitro and in vivo. In present study, the aim is to directly compare umbilical cord blood (UCB)- and BM-derived late EPC surface phenotypes and in vitro functional capacity. Methods: Mononuclear cells from UCB and BM cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Late outgrowing endothelail cells(late OECs) which were in peak growth at third weeks of culture were analyzed for expression of various surface markers by flow cytometry/RT-PCR/IF, tube formation in Matrigel plates, proliferation assay, endothelial colony assay and the role of SDF-1/VEGF on migration. Results: The adherent cells after culture of 7 days exhibited a fibroblast like shape in BM and a cobblestone shaped cells in UCB. Although two sources of OECs were comparable in expression of endothelial and various adhesion molecule markers, BM-derived OECs contained higher proportion of cells expressing smooth muscle cell markers(SMMHC), several adhesion molecule(CD49d, CD62L and VCAM-1), whereas the expression of CXCR-4, PECAM-1 and CD62E and expression of mRNA on endothelial marker genes were higher in UCB-derived OECs. UCB-OECS stained positive for uptake of acetylated low-density lipoprotein and had more migratory ability in the presence of SDF-1 and VEGF compared with BM-OECs. Both sources OECs effectively formed capillary tubes in Matrigel plates. Conclusion: We directly compared OECs derived from UCB and BM and two source of OECs differ in aspect of several adhesion molecule and angiogenic potential in vitro. These difference of UCB render it potentially advantageous for human therapeutic OECs applications for potential applications for a “cell therapy” in the situations on vascular injuries when compared with patients-derived BM.

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Bradykinin Protects Human Endothelial Progenitor Cells from High-Glucose-Induced Senescence through B2 Receptor-Mediated Activation of the Akt/eNOS Signalling Pathway

Journal of Diabetes Research ◽

10.1155/2021/6626627 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Yuehuan Wu ◽

Cong Fu ◽

Bing Li ◽

Chang Liu ◽

Zhi He ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

High Glucose ◽

Copy Number ◽

Relative Length ◽

Healthy Controls ◽

Mtdna Copy Number ◽

Endothelial Progenitor ◽

Cd34 Cells

Background. Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. Methods. Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. Results. B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. Conclusion. BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

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Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells

Experimental and Therapeutic Medicine ◽

10.3892/etm.2014.1827 ◽

2014 ◽

Vol 8 (3) ◽

pp. 805-812 ◽

Cited By ~ 2

Author(s):

JUN-FENG LIU ◽

ZHONG-DONG DU ◽

ZHI CHEN ◽

ZHI-XU HE

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Bone Marrow Cell ◽

Marrow Cell ◽

Endothelial Progenitor ◽

In Vitro Expansion ◽

Bone Marrow Cell Culture

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MRI Tracking of FePro Labeled Fresh and Cryopreserved Long Term In Vitro Expanded Human Cord Blood AC133+ Endothelial Progenitor Cells in Rat Glioma

PLoS ONE ◽

10.1371/journal.pone.0037577 ◽

2012 ◽

Vol 7 (5) ◽

pp. e37577 ◽

Cited By ~ 10

Author(s):

Branislava Janic ◽

Kourosh Jafari-Khouzani ◽

Abbas Babajani-Feremi ◽

A. S. M. Iskander ◽

Nadimpalli Ravi S. Varma ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Endothelial Progenitor ◽

Human Cord Blood ◽

Rat Glioma

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Human Cord Blood-Derived AC133+ Progenitor Cells Preserve Endothelial Progenitor Characteristics after Long Term In Vitro Expansion

PLoS ONE ◽

10.1371/journal.pone.0009173 ◽

2010 ◽

Vol 5 (2) ◽

pp. e9173 ◽

Cited By ~ 38

Author(s):

Branislava Janic ◽

Austin M. Guo ◽

A. S. M. Iskander ◽

Nadimpalli Ravi S. Varma ◽

Alfonso G. Scicli ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor ◽

Human Cord Blood ◽

In Vitro Expansion

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Vessel wall–derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells

Blood ◽

10.1182/blood-2004-08-3057 ◽

2005 ◽

Vol 105 (7) ◽

pp. 2783-2786 ◽

Cited By ~ 397

Author(s):

David A. Ingram ◽

Laura E. Mead ◽

Daniel B. Moore ◽

Wayne Woodard ◽

Amy Fenoglio ◽

...

Keyword(s):

Endothelial Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Ex Vivo ◽

Umbilical Vein ◽

Proliferative Potential ◽

Endothelial Progenitor ◽

Vessel Walls

AbstractEndothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood and expanded exponentially ex vivo. In contrast, human umbilical vein endothelial cells (HUVECs) or human aortic endothelial cells (HAECs) derived from vessel walls are widely considered to be differentiated, mature endothelial cells (ECs). However, similar to adult- and cord blood–derived EPCs, HUVECs and HAECs derived from vessel walls can be passaged for at least 40 population doublings in vitro. Based on this paradox, we tested whether EPCs reside in HUVECs or HAECs utilizing a novel single cell deposition assay that discriminates EPCs based on their proliferative and clonogenic potential. We demonstrate that a complete hierarchy of EPCs can be identified in HUVECs and HAECs derived from vessel walls and discriminated by their clonogenic and proliferative potential. This study provides evidence that a diversity of EPCs exists in human vessels and provides a conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity.

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