Derivation of Functional Endothelial Progenitor Cells from Human Umbilical Cord Blood Mononuclear Cells Isolated by a Novel Cell Filtration Device

Abstract

Endothelial progenitor cells (EPCs) can differentiate from mononuclear cells (MNCs) of adult human peripheral blood, bone marrow, and cord blood during culture. Although MNCs are usually isolated by a Ficoll gradient centrifuge method, this method is time‐consuming, and blood is easily contaminated. We developed a novel cell filtration device (StemQuick™E, Asahi Kasei Medical, Oita, Tokyo, Japan) to isolate MNCs from human cord blood and examined whether functional EPCs could differentiate from MNCs isolated by this device. Recovery rates of MNCs, CD34+ and CD133+ progenitor cells, were significantly greater in the StemQuick™E method than in the Ficoll method. During MNC culture, spindle‐shaped attaching cells developed, and most of these cells incorporated DiI‐acetylated low‐density lipoprotein and showed positive binding to fluorescein isothiocyanate–lectin. Reverse transcription–polymerase chain reaction analysis revealed that attaching cells expressed various progenitor and endothelial lineage markers such as KDR, CD31, endothelial cell nitric oxide synthase, CD133, and LOX‐1. Culture‐expanded EPCs were isolated and labeled with a green fluorescent dye, PKH2‐GL, and cocultured with human umbilical vein endothelial cells (HUVECs). EPCs formed angiogenesis‐like networks together with HUVECs in 3D matrix gel. Finally, EPCs (3 × 105) were implanted into ischemic hindlimb of nude rats (n = 3), and laser Doppler blood flowmetry (LDBF) revealed that the ratio of ischemic to normal limb LDBF was significantly greater in EPC‐transplanted animals compared with controls receiving saline. In conclusion, the novel cell filtration device, StemQuick™E, is a useful tool to isolate MNCs from human cord blood. Moreover, MNCs obtained by this filter system can give rise to functional EPCs.

Therapeutic Angiogenesis With Intramuscular Injection of Low-Dose Recombinant Granulocyte-Colony Stimulating Factor

“…Total RNA was reverse transcribed using oligo dT primers and RNase H reverse-transcriptase (Superscript II; Invitrogen) with 1 g total RNA per sample. 14 The primer set for detecting human G-CSF receptor mRNA was: for (sense) 5Ј-TCCTCAC-CCTGATGACCTTGA-3Ј, and for (antisense) 5Ј-GAAGTTGAGC-AGTGGCCCAAA-3Ј. RT-PCR of G-CSF receptor was expected to yield 3 PCR products sized 620 bp (transmembrane form, GenBank M59818), 701 bp (insert in the cytoplasmic domain, GenBank M59820), and 548 bp (soluble form, GenBank M59819).…”

A Systematic Approach to the Establishment and Characterization of Endothelial Progenitor Cells for Gene Therapy

“…Because of the lack of an EPC-specific cell surface marker, the phenotype of adherent cells was further characterized using dual labeling of the cells with acetylated low-density lipoprotein (AcLDL) and UEA-1 lectin, an additional indication of EPC-like properties (Aoki et al, 2004). More than 85% of early EPCs (CD34 + CD31 + CD45 + ) from both WBC filters and buffy coat blood showed dual-labeling of AcLDL and UEA-1 lectin (Fig.…”

Current data on predictive markers for anti-angiogenic therapy in thoracic tumours

“…Also, results generated by flow cytometry analyses used by most authors may be influenced by several factors, such as separation of mononuclear cells, washing and centrifugation steps, the panel of monoclonal antibodies and the lack of standardisation of gating techniques. Hence, various improvements have been attempted, including specific and nonspecific antibody combinations [88] and isolation by size using a defined filter for isolation of EPCs instead of centrifugation of blood samples [89].…”