Simultaneous Isolation of Human Bone Marrow Hematopoietic, Endothelial, and Mesenchymal Progenitor Cells by Flow Sorting Based on Aldehyde Dehydrogenase Activity.
Authors: Gentry Tracy , Foster Sandra J. , Winstead Lisa , Deibert Erica , Fiordalisi Michelle , Balber Andrew E. 
Published: October 2019

Abstract
ALDH bright [ALDHbr] cells exhibit low side scatter and high activity of the intracellular enzyme aldehyde dehydrogenase [ALDH] in flow cytometric analysis. ALDH regulates gene expression in many developing systems by producing retinoic acids. ALDHbr populations from human umbilical cord [UCB] and mobilized peripheral blood preparations [PBSC] are highly enriched for cells that express CD34 and CD133 and for hematopoeitic progenitor cells. Because human bone marrow [BM] contains a highly heterogeneous population of stem and progenitor cells, we used multiparameter flow analysis and cell sorting to measure expression of cell surface markers and lineage specific developmental potential of BM ALDHbr populations. The ALDHbr population comprised 1.2 ± 0.8% (mean ± SD, n=39) of the nucleated BM cells and was significantly (p<0.05) enriched in cells expressing CD34, CD117, CD105, CD127, CD133, and CD166. ALDHbr populations were also significantly enriched in cells expressing primitive progenitor phenotypes including CD34+CD38− and CD34+CD133+ cells. The cell surface antigen expression of BM ALDHbr populations differed significantly from UCB and PBSC populations. In particular, the BM ALDHbr populations had more CD34− cells and CD45− cells and fewer CD133+ cells than the UCB and PBSC ALDHbr populations. Next, we compared the ability of sorted ALDHbr cells and ALDHdim cells derived from each bone marrow to develop into erythroid and myeloid cells, megakaryocytes, endothelial cells, and mesenchymal cells in culture. The ALDHdim population was the residual cell population produced when we sorted ALDHbr cells out of BM. ALDHbr populations were 144-fold more active in primary hematopoietic colony forming activity than ALDHdim cells, and all detectable BM cells that could form megakaryocyte colonies were ALDHbr. Even though hematopoietic progenitor activity was concentrated in the ALDHbr population, the majority of CD34+ and CD133+ cells in BM were ALDHdim. Cultures seeded with 50,000 ALDHbr cells gave rise to confluent growth of endothelial cells that expressed surface Ulex lectin receptors and von Willebrand factor, formed tubules on Matrigel, and took up acetylated LDL in 34 of 39 BM tested. In contrast, only 18 of 33 ALDHdim BM populations tested gave rise to endothelial cultures, and 500,000 ALDHdim cells were required to generate confluent cultures in 21 days. Cells that gave rise to early endothelial growth colonies at day 7 were 435-fold more frequent in the ALDHbr than the ALDHdim population. Confluent cultures of cells expressing cell surface antigens and functional attributes of multipotential mesenchymal cells were also routinely established with 50,000 ALDHbr cells. Again, ten-fold more ALDHdim cells than ALDHbr cells were needed to establish mesenchymal cultures. The mesenchymal cultures could be driven to differentiate into adipocytes, chondrocytes, osteoblasts, and neural lineage cells in standard culture assays. These results show that most of the hematopoietic, endothelial, and mesenchymal progenitor cell activities in human BM can be simultaneously isolated by cell sorting based on ALDH activity. Such ALDHbr populations may be useful in a variety of cell therapy indications.