231ABC-Transporter mediated efflux of aldehyde dehydrogenase reaction product: Implications for enumeration and isolation of functional blood progenitor cells

Identification of stem cells based on hematopoietic stem cell (HSC) surface markers, such as stem cell antigen-1 (Sca-1) and the c-kit receptor, has limited specificity. High aldehyde-dehydrogenase (ALDH) activity is a general cellular property of stem cells shared by HSC, neural, and intestinal stem cells. The presence of cells with high ALDH activity in the adult heart has not been investigated. Methods: Cells were isolated from adult mouse hearts, and from atrial appendage samples from humans with ischemic or valvular heart disease. Myocyte-depleted mouse Sca-1+, and lineage (Lin)-negative/c-kit+ human heart cells were purified with immunomagnetic beads. ALDH-high cells were identified using a specific fluorescent substrate, and sorted by FACS. Cell surface marker analysis was performed by flow cytometry. Results: Myocyte-depleted mouse heart cells contained 4.8+/−3.2% ALDH-high/SSC-low and 32.6+/−1.6% Sca-1+ cells. ALDH-high cells were Lin-negative, Sca-1+ CD34+ CD105+ CD106+, contained small CD44+ (27%) and CD45+ (15%) subpopulations, and were essentially negative for c-kit (2%), CD29, CD31, CD133 and Flk-1. After several passages in culture, ~20% of ALDH-high cells remained ALDH-high. Myocyte-depleted human atrial cells contained variable numbers of ALDH-high cells ranging from 0.5% to 11%, and 4% Lin-negative/c-kit+ cells. ALDH-high cells were CD29+ CD105+, contained a small c-kit+ subpopulation (5%), and were negative for CD31, CD45 and CD133. After 5 passages in culture, the majority of ALDH-high cells remained ALDH-high. Conclusions: Adult mouse and human hearts contain significant numbers of cells with high ALDH activity, a general cellular property that stem cells possess in different organs, and express stem cell markers (Sca-1 and CD34 in the mouse). The immunophenotype of cardiac-resident ALDH-high cells differs from that previously described for bone marrow ALDH-high HSC, and suggests that this cell population may be enriched in mesenchymal progenitors. Analysis of lineage differentiation potential of ALDH-high cells is in progress. ALDH activity provides a new, practical approach to purifying cardiac-resident progenitor cells.

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Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy

Cytotherapy ◽

10.1080/14653240701218516 ◽

2007 ◽

Vol 9 (3) ◽

pp. 259-274 ◽

Cited By ~ 81

Author(s):

T. Gentry ◽

S. Foster ◽

L. Winstead ◽

E. Deibert ◽

M. Fiordalisi ◽

...

Keyword(s):

Cell Therapy ◽

Progenitor Cells ◽

Dehydrogenase Activity ◽

Aldehyde Dehydrogenase ◽

Mesenchymal Progenitor Cells ◽

Flow Sorting ◽

Aldehyde Dehydrogenase Activity

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Human mitochondrial aldehyde dehydrogenase substrate specificity: Comparison of esterase with dehydrogenase reaction

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(92)90239-s ◽

1992 ◽

Vol 299 (1) ◽

pp. 23-29 ◽

Cited By ~ 24

Author(s):

Neeta Mukerjee ◽

Regina Pietruszko

Keyword(s):

Substrate Specificity ◽

Aldehyde Dehydrogenase ◽

Dehydrogenase Reaction

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CD133-Expressing Cells with High Aldehyde Dehydrogenase Activity Represent a Primitive Human Hematopoietic Stem Cell Population with Enhanced Repopulating Potential.

Blood ◽

10.1182/blood.v104.11.250.250 ◽

2004 ◽

Vol 104 (11) ◽

pp. 250-250

Author(s):

David A. Hess ◽

Louisa Wirthlin ◽

Timothy P. Craft ◽

Todd E. Meyerrose ◽

Jan A. Nolta

Keyword(s):

Progenitor Cells ◽

Aldehyde Dehydrogenase ◽

Mononuclear Cells ◽

Phenotypic Expression ◽

Stem Cell Population ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Aldh Activity ◽

Hematopoietic Stem ◽

Selection Of

Abstract Human hematopoietic stem cells (HSC) are commonly purified by the phenotypic expression of cell surface markers such as CD34. We have recently characterized a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Lin− cells with high ALDH activity (ALDHhiLin−) represented approximately 0.1% of total UCB mononuclear cells and demonstrated enriched expression of the primitive HSC markers CD34 (91.0±2.9%) and CD133 (70.9±4.0%). Most notably, clonogenic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin− population. Here, we have further purified the ALDHhiLin− population based on the expression of CD133, or prominen, a non-restricted surface molecule expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133− and ALDHhiCD133+ cells, sorted to >95% purity, represented 14.7±2.1% and 23.2±4.3% of the total human UCB Lin− population respectively (n=6). Both ALDHhiCD133−Lin− and ALDHhiCD133+Lin− cells demonstrated clonogenic progenitor function in vitro. However, total colony production was significantly enhanced (p<0.05) in ALDHhiCD133−Lin− cells (1 CFU in 3.5 cells, n=5) when directly compared to ALDHhiCD133+Lin− cells (1 CFU in 10 cells, n=6). Human hematopoietic repopulation was consistently observed in the bone marrow, spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 103 ALDHhiCD133+Lin− cells, whereas transplantation of up to 2x105 ALDHhiCD133−Lin− cells produced no detectable human engraftment. BM repopulation at limiting dilution demonstrated increased NOD/SCID repopulating ability elicited by ALDHhiCD133+Lin− cells when directly compared to CD133+Lin− cells not selected for ALDH activity. Repopulating ALDHhiCD133+Lin− cells differentiated into cells expressing markers for mature myeloid (CD33, CD14) and B-lymphoid (CD19, CD20) cells. ALDHhiCD133+Lin− cells also supported the maintenance of primitive cell phenotypes up to 8 weeks post-transplantation (2.4±0.7% CD34+CD38−, 5.5±0.6% CD34+CD133+, n=6) and the repopulating function of these cells are currently being confirmed by secondary transplantation. We are also investigating the ability of ALDHhiCD133+Lin− cells to mediate tissue repair in non-hematopoietic organs. Fractionation of human HSC based on combined expression of CD133 and high ALDH activity provides a rigorous selection of purified hematopoietic stem and progenitor cells that maintain primitive characteristics after transplantation, and may be considered a potential alternative to CD34+ cell isolation.

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