Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity

Abstract

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells, 40-90% pure for CD34(+)CD38(lo/-) cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.

Figure 1

Structures of BAAA-DA, BAAA, and BAA. BAAA-DA was synthesized by the bonding of the fluorescent compound BODIPY FL,SE to AAA-DA. BAAA-DA is converted to BAAA in the presence of acid with a t_1/2 of approximately 15 min under the conditions described in Materials and Methods. When cells are stained with BAAA, ALDH then converts BAAA into BAA, which is retained intracellularly because of its net negative charge. The structures of BAAA-DA and BAAA as well as the kinetics of conversion between these compounds were determined by ¹H-NMR spectroscopy.

Figure 2

BAAA staining of L1210, L1210/cpa, and K562 cells. L1210 (A) is a murine cell line that expresses little ALDH. L1210/cpa (B) expresses high levels of ALDH, and K562 (C) is a human cell line that expresses ALDH. Cells were stained with 20 μM BAAA and analyzed as described in Materials and Methods. The data are representative of three experiments.

Figure 3

Human UCB contains ALDH^br cells. Red cell-depleted UCB mononuclear cells were stained with 1 μM BAAA. (A) The scatter properties of stained UCB. Region R1 defines the SSC^lo population and R2 defines the monocyte population. (B–D) The BAAA staining profile of the total UCB, monocytes, and SSC^lo cells, respectively. The SSC^loALDH^br cells are defined by the M1 cursor in D. In each panel the dotted line is the diethylaminobenzaldehyde-treated negative control and the solid line is the experimental sample. The data are representative of six experiments.

Figure 4

SSC^loALDH^br UCB is enriched for CD34⁺CD38^lo/− cells. UCB was costained with BAAA, verapamil, and antibodies to CD34 and CD38 and analyzed with FACS. (A) The CD34 and CD38 staining profile of unfractionated UCB. (B) The profile of SSC^lo cells. (C) The profile of SSC^loALDH^br cells. The data are representative of six experiments.

Figure 5

SSC^loALDH^br UCB is enriched in hematopoietic progenitors. Unfractionated, CD34⁺, and SSC^loALDH^br UCB cells were isolated and placed into HPCA (A), 5-week LTC (B), and 8-week LTC (C) assays. As described in Materials and Methods, CD14⁺ cells also were removed during the isolation of the SSC^loALDH^br population because these cells were the major contaminant in preliminary sorting experiments. The bar graphs demonstrate the mean (±SD) of six experiments. The difference in HPCA, 5-week LTC, and 8-week LTC was significantly different (P < 0.05) between unfractionated UCB and both the CD34⁺ and SSC^loALDH^br populations. In addition, the proportion of 8-week LTCs was significantly higher (P < 0.05) in the SSC^loALDH^br population compared with the CD34⁺ population whereas no differences were noted between the HPCA and 5-week LTC proportions.