Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity

Abstract

IntroductionConventionally, human hematopoietic stem cells (HSCs) have been purified on the basis of the expression of cell surface molecules such as CD34 and CD133. [1][2][3][4][5] Cells expressing CD34 are capable of long-term hematopoietic reconstitution in immune-deficient mice and fetal sheep, [1][2][3]6,7 and most clinical protocols involving gene transfer, purified stem cell transplantation, and stem cell expansion have been designed with the use of CD34 ϩ populations. [8][9][10] However, findings in a variety of mammalian systems suggest that the human hematopoietic stem cell compartment is heterogeneous, including an array of cell phenotypes that exhibit repopulating function. [11][12][13][14] In fact, human CD34 ϩ and CD34 Ϫ populations possess repopulating ability, and CD34 expression is reversible in mouse and human cells. [15][16][17][18][19] Although CD34 selection has proven extremely useful for the isolation and characterization of human cells with enhanced repopulating function, there are several limitations to purifying hematopoietic cells based solely on cell surface phenotype. First, cell surface markers can vary between species and stem cell source. Murine long-term repopulating cells have been purified to a single cell with the use of CD34 Ϫ , c-kit ϩ , Sca-1 ϩ , Lin Ϫ cell markers, 11 whereas identification of a human single cell phenotype capable of repopulating murine or fetal sheep xenograft models remains elusive. 1,12,14 Second, human stem cell phenotype can be altered by cell cycle progression, 20,21 ex vivo culture, 14,19,22 and transplantation in vivo, 12,15,16 indicating a dissociation between hematopoietic cell phenotype and repopulating cell function. Third, the phenotype of transplanted stem cells may remain constant despite reduced functional activity. Cytokinemobilized peripheral blood cells demonstrate increased CD34 expression without an increase in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating ability. 23,24 Finally, purification of human stem cells on the basis of hematopoietic markers may select cells with restricted developmental potential and may exclude cells with alternate stem cell or progenitor functions. Thus, methods to efficiently isolate human stem cells without relying entirely on phenotypic cell surface molecules are desirable.One promising strategy is HSC isolation according to a conserved stem cell function rather than phenotype. In the murine system, lymphohematopoietic stem cells have been isolated according to the high expression of the detoxifying enzyme aldehyde dehydrogenase (ALDH). [25][26][27] Cytosolic ALDH is also expressed at high levels in human hematopoietic progenitors, 28 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-proprionic acid) fluorochrome that is metabolized by ALDH to an aminoacetate anion, retained within the cell because of its negative charge. Thus, the amount of fluorescent product that accumulates in viable cells correlates to ALDH activity, and cells with high ALDH activity can be selected from...

Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration

“…Within 24 h, mononuclear cells were isolated by Ficoll-Hypaque centrifugation, and cells with low ALDH activity (ALDH lo ), as well as ALDH hi cells, were purified by cell sorting based on ALDH activity using Aldefluor reagent (Stem Cell Technologies, Vancouver, BC, Canada) as previously described [6]. ALDH lo and ALDH hi cells were characterised for mature haematopoietic and primitive progenitor marker expression, and colonyforming cell (CFC) assays were performed for haematopoietic, endothelial and multipotent stromal progenitor functions as previously described [7].…”

Cord blood banking for clinical transplantation

“…Most important for the transplant recipient, however, is the presence of sufficient numbers of viable stem and progenitor cells in CBUs, more closely reflected by the counts of mononuclear cell (MNC) and CFUs 30 and CD34 þ cells. 31 Other tests of hematopoietic potency have been proposed, the expression of aldehyde dehydrogenase by MNCs 32 and the levels of ATP after culture in the presence of selective cytokine cocktails. 33 Information on the clinical relevance of these reasonably putative markers is still preliminary.…”

Dual SP/ALDH Functionalities Refine the Human Hematopoietic Lin−CD34+CD38− Stem/Progenitor Cell Compartment

“…It is generally held that the SP compartment involves only a small fraction of CD34 þ cells, in contrast to the ALDH Br population, which is mainly composed of CD34 þ cells (10.67% AE 13.1% and 91.0% AE 2.9%, respectively) [23,24]. In our experiments, the percentages of CD34 þ cells in the Lin À SSC Low SP and Lin À SSC Low ALDH Br fractions were 72.6% AE 16.2% and 91.2% AE 13.1%, respectively.…”

“…Similar to the SP phenotype, the ALDH activity level is considered as a stem cell marker because it is expressed in stem cells from several tissues, including hematopoietic, keratocytic, mesenchymal, neuronal, and endothelial stem cells [21,22]. Whereas the cell fraction showing the highest expression of ALDH (ALDH Br ) contains the largest number of CD34 þ CD38 Low/À HSCs/HPCs [18,23], it also includes primitive CD34 À HSCs/HPCs [24], suggesting that, similar to SP cells, the ALDH Br compartment is heterogeneous, at least phenotypically.…”