Isolation of Human Umbilical Cord Blood Aldehyde Dehydrogenase–Expressing Progenitor Cells that Modulate Vascular Regenerative Functions In Vitro and In Vivo

Abstract

This unit describes the isolation and application of human umbilical cord blood progenitor cells to modulate vascular regenerative functions using in vitro co‐culture systems and in vivo transplantation models. Using aldehyde dehydrogenase as a marker of stem cell function, blood‐derived progenitors can be efficiently purified form human umbilical cord blood using flow cytometry. We describe in vitro approaches to measure cell‐mediated effects on the survival, proliferation, and tube‐forming function of endothelial cells using growth‐rate assays and Matrigel tube‐forming assays. Additionally, we provide a detailed protocol for inducing acute unilateral hindlimb ischemia in immune‐deficient mice to assess progenitor cell–modulated effects on vascular regeneration by tracking the recovery of blood flow using noninvasive laser Doppler perfusion imaging. Collectively, we present combined in vitro and in vivo transplantation strategies for the pre‐clinical assessment of human progenitor cell–based therapies to treat ischemic disease. Curr. Protoc. Stem Cell Biol. 25:2A.10.1‐2A.10.19. © 2013 by John Wiley & Sons, Inc.

High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential

“…At passage 4 (P4), MSCs were purified into ALDH l ° versus ALDH hi subsets by fluorescence activated cell sorting (FACS) using the Aldefluor assay (StemCell Technologies, Vancouver, BC, http://www.stemcell.com) as described previously . The ALDH hi subset represented cells with approximately Five‐fold higher fluorescence intensity compared with ALDH l ° gate established using DEAB‐inhibition.…”

“…After 14 or 21 days, cells were fixed in formalin and stained for adipocytes or osteocytes using using oil red O or Alizarin red, respectively. For chondrogenic differentiation, micromasses of purified ALDH l ° or ALDH hi MSCs ( N = 3) were cultured for 14 days in chondrogenesis differentiation media (Life Technologies) as described previously . Micromasses were frozen in optimal cutting temperature (OCT), sectioned, and stained with Alcian Blue counterstained with Nuclear Fast Red.…”

Concise Review: Cell Therapy for Critical Limb Ischemia: An Integrated Review of Preclinical and Clinical Studies

“…Since Asahara et al first discovered circulating EPC in 1997 , the regeneration of blood vessels using stem cells has undergone intense preclinical investigation . The surgical hindlimb ischemia model performed by femoral artery ligation in immunodeficient mice has been used for proof‐of‐concept to establish the efficacy of transplanted human cell populations . Cells were usually administered by intravenous or multiple IM injections proximal to the ligation site, blood flow was monitored noninvasively by laser Doppler perfusion imaging, and vessel formation was quantified in muscle using a battery of immunohistochemical stains.…”

Aldehyde dehydrogenases in acute myeloid leukemia

“…In addition, measuring these cells in stored or shipped PBSC, BM, and UCB grafts may be a useful quality‐assurance assessment of viable HSCs and progenitors that have survived these manipulations . Additionally, CD45 − ALDH br cells in the marrow and blood may be enriched for mesenchymal stem cells (MSC) and endothelial progenitors cells, both of which may play important roles in the HSC and hematopoietic progenitor environmental niche . Clinical trials are underway using cells sorted from BM on the basis of ALDH activity to repair cardiac and brain tissue damaged by myocardial infarction and stroke, respectively …”