Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Abstract

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues. Later, others documented the existence of similar types of EPCs originating from bone marrow. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

TWIST1-Reprogrammed Endothelial Cell Transplantation Potentiates Neovascularization-Mediated Diabetic Wound Tissue Regeneration

“…However, prior literature suggests that transplantation of mature EC fails to improve vascularity as well as functional improvement of the injured tissue (37,38). Recent studies on the clinical and therapeutic application of autologous EPC transplantation depicted limited improvement in neovascularization and tissue repair (39). A reduced levels as well as dysfunctional EC with an impaired proliferative, and mobilization capability, integrin profile, low differentiation and incorporation into new blood vessels were observed in Type 1 and Type 2 diabetes (40-42) thereby, limiting the source of EPC to be autologous in origin for transplantation.…”

Endothelial Progenitor Cells: An Appraisal of Relevant Data from Bench to Bedside

“…In fact, some CD34+/CD133+/VEGFR-2+ cells are positive for the common leukocyte marker CD45 and therefore are likely to represent hematopoietic rather than endothelial precursors [ 18 ]. On the other hand, FACS is a relatively sensitive technique but with rapid advancement in technology; instead of MACS, FACS has not only gained significant attention but is now the mainstay to isolate, classify, and analyse progenitor cells because of its versatility and ease in obtaining a high percentage of pure populations [ 19 , 20 ].…”

Células madre mesenquimatosas y sus diferentes usos

“…Por último, las células adheridas deben ser separadas, lo cual se realiza con tripsina o colagenasa. 20,21 Para la visualización de su morfología, se usa un microscopio electrónico de barrido y emisión para poder evaluar la viabilidad que estas potencialmente tendrían. 22 Para asegurar esa viabilidad se implementa una coloración con calceína-ester de acetoximetilo y homodímero de etidio que permite visualizar en un microscopio de barrido de laser focal la cantidad de fluorescencia de las células, la cual se relaciona de manera directamente proporcional con la viabilidad de las mismas.…”

“…23 Finalmente, para determinar el fenotipo células se realiza una citometría de flujo o, en su defecto, un western blot para identificar y aislar las células correspondientes. 20,21 Por medio de la citometría de flujo se evidencia la presencia o ausencia de marcadores que permite asegurar que las células recolectadas son CMM con la facultad de diferenciarse a múltiples tejidos entre ellos adipocitos, osteocitos, condrocitos, cardiomiocitos, entre otros. 3…”