Phenotypic Characterization of Murine Primitive Hematopoietic Progenitor Cells Isolated on Basis of Aldehyde Dehydrogenase Activity
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《干细胞学杂志》
a Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Newcastle upon Tyne, United Kingdom;
b BD Biosciences, Oxford, United Kingdom;
c Department of Biological Sciences, University of Durham, Durham, United Kingdom
Key Words. Hematopoietic stem cells ? Aldehyde dehydrogenase activity ? Telomerase activity ? Murine hematopoietic progenitor cells
Correspondence: Majlinda Lako, Ph.D., Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00-44-191-241-8688; Fax: 00-44-191-241-8666; e-mail: Majlinda.Lako@ncl.ac.uk
ABSTRACT
Hematopoietic stem cells (HSCs) are multipotent cells that are normally resident in the bone marrow and are responsible for producing all of the adult hematopoietic lineages. They can be enriched from bone marrow and used for transplantation purposes, and the ease of accessibility of the bone marrow makes them an attractive model system in which to investigate the stem cell biology of renewing tissues. Most enrichment procedures for HSCs have involved the fractionation of cells based on the expression of cell-surface antigens or functional characteristics of the cells such as low-intensity fluorescence of Rhodamine 123 or Hoechst 33342 staining . There are several problems associated with these techniques. Primarily, the isolated populations, although highly enriched, still contain significant numbers of more mature progenitor cells . Second, the expression of surface antigens has been shown to vary among species, genetic background, and stem cell source . Some of these techniques require excitation at 350 nm (UV range), which may be harmful to the target cells. These procedures can be technically difficult and do not readily lend themselves to clinical applications. It is desirable, therefore, to develop alternative methods by which stem cell isolation may be achieved.
The aldehyde dehydrogenases (ALDHs) are intracellular enzymes responsible for oxidizing aldehydes to carboxylic acids . Members of this class are central to the processing of ethanol and amines produced during the catabolism of catecholamines and the conversion of vitamin A to retinoic acid . Both the hematopoietic progenitor cells and intestinal crypt cells display high levels of cytosolic ALDH and are relatively resistant to cyclophosphamide . To make use of this feature for purification of HSCs, several groups have developed fluorescent substrates for these enzymes. The first of these was based on the oxidation of dansylaminoacetaldehyde to dansylglycine . Jones et al. used this substrate to purify viable hematopoietic progenitors that were able to confer delayed but stable engraftment of myeloablated recipients and lacked characteristic cell-surface HSC markers. This substrate suffers from the disadvantage of UV excitation, which could be mutagenic to the cells, and the emission spectrum of this substrate overlaps with other fluorochromes and cannot readily be combined with other stem cell markers. In 1996, Storms et al. devised a new system that uses a visible light excitable fluorophore (BODIPY-conjugated aminoacetaldehyde) that is metabolized by ALDH to a carboxylate ion retained within the cell, allowing cells with high levels of ALDH to be isolated by fluorescence-activated cell sorter (FACS) because of their high fluorescence. Using a two-step strategy that combines the depletion of cells that express mature lineage markers with high activity of ALDH, highly purified hematopoietic-repopulating cells were purified from human umbilical cord blood using the BODIPY-based substrate named Aldefluor. This substrate had not been applied to murine models, and we wished therefore to investigate its application for the purification of murine hematopoietic progenitor from bone marrow. Using lineage depletion followed by Aldefluor staining, we were able to isolate a highly purified population of hematopoietic progenitors from murine bone marrow of both C57BL6 and BALB/c mice. Furthermore, we have shown that Lin–ALDHbright cells show cell-surface marker characteristics of primitive hematopoietic progenitors and are highly quiescent and enriched for hematopoietic progenitor activity.
MATERIALS AND METHODS
Purification of Lin–ALDHbright and Lin–ALDHlow Cells
To enrich for hematopoietic progenitors, we carried out a lineage depletion of committed cells using anti-PE magnetic beads (Miltenyi Biotech) and antibodies directed against lymphoid, myeloid, and erythroid cells. The N,N-diethyl-aminobenzaldehyde inhibitor sample was analyzed first using FL1 and side scatter suggested by the manufacturer, and the R1 gate was chosen to represent the background level of cytosolic ALDH activity (Lin–ALDHlow; Fig. 1A). Staining of Lin– cells with Aldefluor but without the inhibitor caused a shift in fluorescence, which allowed us to define the Lin–ALDHbright cells in gate R2 (Fig. 1B). The R2 region comprised 26.8 ± 1.0% of the Lin– population in C57BL6 mice and 0.058 ± 0.01% of the total bone marrow cells (data derived from seven independent experiments). Similarly, in BALB/c mice, the R2 region contained 23.5 ± 1.0% of the Lin– population and 0.055 ± 0.01% of the total population (data derived from three independent experiments; supplementary online Fig. 1B).
Figure 1. Purification of Lin–ALDHbright and Lin–ALDHlow cell populations in C57BL6 mice. Murine bone marrow cells were subjected to lineage depletion before staining with Aldefluor substrate. (A): Lin– cells stained with Aldefluor substrate and DEAB inhibitor were analyzed first to establish the baseline level of ALDH activity (gate R1, Lin–ALDHlow cell population); a dot plot and analysis contour plot are shown on the left and right sides, respectively. (B): Staining of Lin– cells with Aldefluor but without DEAB inhibitor produced a shift in fluorescence that defined the Lin–ALDHbright population (R2 gate; 26.8 ± 1.0% of the Lin– population); a dot plot and analysis contour plot are shown on the left and right sides, respectively. The data represent the mean ± standard error of the mean performed on seven independent Lin– cells purified from murine bone marrow. Abbreviations: ALDH, aldehyde dehydrogenase; DEAB, N,N-diethylaminobenzaldehyde.
Characterization of Lin–ALDHbright and Lin–ALDHlow Cells
Phenotypic investigation of Lin–ALDHbright cells in C57BL6 mice was performed using cell-surface markers common to the hematopoietic lineage (CD45; Figs. 2A, 2B), hematopoietic stem cell markers (c-kit, CD34, Sca-1, Flk-2, CD38, CD44; Figs. 2C–2J, 2M–2P), endothelial progenitor marker, Flk-1 (Figs. 2K, 2L), and mesenchymal stem cell markers (CD13, CD81, CD29; Figs. 2Q–2V). The Lin–ALDHbright population was comprised entirely of hematopoietic cells and devoid of mesenchymal or endothelial progenitors, as shown by specific marker staining. In addition, the Lin–ALDHbright population contained a higher percentage of cells that expressed the HSC marker c-kit compared with Lin–ALDHlow cells (Figs. 2C, 2D) but lacked expression of Sca-1 (Figs. 2G, 2H). Lin–ALDHbright cells were mostly devoid of CD34 (Figs. 2I, 2J) and CD38 expression (Figs. 2M, 2N), as demonstrated previously for murine hematopoietic stem cells in newborn and juvenile mice . Higuchi et al. have reported that a CD38+ subpopulation of HSCs appears before the age of 5 weeks and expands during adolescence. To ensure that we did not miss the developmental time point at which CD38 expression is observed in murine HSCs, we repeated the CD38 phenotypic analysis of Lin–ALDHbright at weeks 12, 14, and 16 (data not shown). We noticed no change in CD38 expression in the Lin–ALDHbright population between 8–12 weeks of development and 16 weeks, suggesting no expansion in CD38+ progenitors in the Lin–ALDHbright during this interval. Because CD38 expression is noticeable upon activation of juvenile HSCs in mice, it is tempting to speculate that the hematopoietic progenitors marked by high levels of ALDH activity are quiescent and might gain CD38 expression upon activation, an investigation that is now underway in our laboratory.
Figure 2. Phenotypic characterization of Lin–ALDHbright and Lin–ALDHlow cell populations in C57BL6 mice. Lin–ALDHbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and analyzed for the expression of CD45 (A, B), c-kit (C, D), Flk-2 (E, F), Sca-1 (G, H), CD34 (I, J), Flk-1 (K, L), CD38 (M, N), CD44 (O, P), CD29 (Q, R), CD81 (S, T), and CD13 (U, V). Data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. Statistical significance of the change between Lin–ALDHbright and Lin–ALDHlow cell populations was calculated using Student’s t-test and is shown as p value for each antibody staining.
All Lin–ALDHbright cells expressed CD44 (Figs. 2O, 2P), a receptor for the cell-surface adhesion molecule hyaluronic acid, which has been shown to be synthesized by primitive hematopoietic cells and is involved in their lodgment to the bone marrow and their proliferation . The classical definition of HSC phenotype in mice includes low expression of Thy-1.1 and high expression of Sca-1; however, this is strain dependent and applicable only to mice expressing the Ly-6A/E and Thy-1.1 haplotypes . C57BL6/J mice express the Thy-1.2 haplotype, and although it has been shown that Thy1.2+ c-kit+ Sca-1+ Lin–/low cells are HSCs, the level of Thy1.2 expression is lower than Thy-1.1, causing Thy1.2+ cells to bleed into the negative gate. In view of these results, it was suggested recently that HSCs from Thy1.2 strains can be isolated by adding Flk-2 to the lineage cocktail . In addition, the loss of Thy-1.1 expression and gain of Flk-2 has been correlated to loss of self-renewal during HSC maturation, such that HSCs lacking Flk-2 have long-term repopulation ability, whereas HSCs with Flk-2 expression show short-term repopulating capacity . Our study showed that Lin–ALDHbright has fewer Flk-2+ cells compared with Lin–ALDHlow (Figs. 2E, 2F), suggesting that although separation on the basis of ALDH activity enriches for primitive progenitor cells, it is likely to include cells with both short- and long-term repopulating ability.
We repeated the same phenotypic analysis on BALB/c mice, which are of different genetic background to C57BL6 mice. We found that Lin–ALDHbright cells were comprised entirely of hematopoietic cells and mostly devoid of mesenchymal or endothelial cells (supplementary online Figs. 2A, 2B, 2M, 2N, 2T–2Y), as were Lin–ALDHbright cells in C57BL6 mice. Lin–ALDHbright cells showed expression of typical HSC markers, such as c-kit and CD44 (supplementary online Figs. 2C–2D, 2R–2S), and mostly lacked CD34 and CD38 expression (supplementary online Figs. 2K, 2L, 2P, 2Q). We did find very low expression of Sca-1 in both Lin–ALDHbright and Lin–ALDHlow cells (supplementary online Figs. 2I, 2J), and this is consistent with previous findings reported by Spangrude and Brooks for the Lin– of BALB/c mice. We found a small percentage of Flk-2+ cells in the Lin–ALDHbright cells (supplementary online Figs. 2E, 2F), suggesting the presence of short-term repopulating cells in the Lin–ALDHbright cells. This analysis showed us that despite genetic background, Lin–ALDHbright cells show phenotypic similarities for the main HSC markers such as c-kit, CD34, CD38, and CD44.
Characterization of Lin–ALDHbright and Lin–ALDHlow Using In Vitro Colony Assays
Cell-surface staining predicted that the Lin–ALDHbright population is likely to be enriched for primitive hematopoietic progenitors. To investigate this possibility, we carried out in vitro colony-forming assays, which allowed us to estimate the plating efficiency of the cells and the nature of progenitors (Fig. 3, Table 1; supplementary online Figs. 3A, 3B). Comparison of the Lin–ALDHbright and Lin–ALDHlow populations showed that the Lin–ALDHbright contained all of the hematopoietic progenitor activity of Lin– cells in both C57BL6 and BABL/C mice, as demonstrated by colony assays (Fig. 3; supplementary online Fig. 3A) and an increased plating efficiency compared with Lin–ALDHlow and Lin– cells (Table 1; supplementary online Fig. 3B). Recent findings have also shown that the Lin–ALDHbright population of umbilical cord blood contains all of the hematopoietic progenitor activity and provides a better tool for resolving the functional differences in repopulating ability between human CD34+ cells . Despite the presence of c-kit expression, Lin–ALDHlow cells did not show any hematopoietic progenitor activity, which indicates that there are functional differences within the c-kit population that can be resolved on the basis of ALDH activity.
Figure 3. Lin–ALDHbright cell population of C57BL6 mice is enriched for hematopoietic progenitor activity. Lin–ALDHbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and subsequently sorted on a BD FACSVantage. In vitro colony assays were performed with Lin–, Lin–ALDHbright, and Lin–ALDHlow cell populations to define the progenitor activity and the plating efficiency of these cells. Data represent the number of individual colonies produced per 100 cells and represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow.
Table 1. Number of colonies per 100 cells
Cell Cycle Status of Lin–ALDHbright and Lin–ALDHlow Cells
Various studies have shown that murine HSCs enter the cell cycle less frequently . We performed cell cycle analysis on Lin–ALDHbright and Lin–ALDHlow cells of C57BL6 mice and found that the Lin–ALDHbright has the lowest content of cycling cells, averaging 10.8 ± 0.7% (Fig. 4A) compared with the Lin–ALDHlow population, which contained 41.5 ± 1.0% of cells in S phase (p = .002, n = 3). Similar results were obtained with the Lin–ALDHbright and Lin–ALDHlow cells of BALB/c mice (supplementary online Fig. 4A). This is consistent with the prediction that these cells contain more primitive progenitors that are quiescent or at least do not contribute to hematopoiesis at any given time .
Figure 4. Cell cycle analysis and telomerase activity of Lin–ALDHbright and Lin–ALDHlow populations of C57BL6 mice. (A): Lin–ALD-Hbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and subsequently sorted on a BD FACSVantage. Phosphatidylinositol staining was used to define the DNA content of the two populations. The data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. (B): Telomerase activity of Lin–ALDHbright and Lin–ALDHlow cell populations was measured using telomeric repeat amplification protocol assays and 1 ng of protein extract as described in Materials and Methods. Data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. Statistical significance of the change between Lin–ALDHbright and Lin–ALDHlow cell populations was calculated using Student’s t-test.
Lin–ALDHbright Cells Have Higher Telomerase Activity Than Lin–ALDHlow Cells
Most cycling HSCs display telomerase activity, and this decreases as HSCs proliferate and differentiate into more mature cells that display negligible levels of this enzyme . To measure telomerase activity, we performed TRAP assays under linear conditions of product amplification using a range of dilutions of protein extracts that we have previously demonstrated to be a suitable range for cells with relatively high telomerase activity . We found that Lin–ALDHbright cells of both C57BL6 and BALB/c mice have significantly higher levels of telomerase compared with Lin–cells and Lin–ALDHlow (Fig. 4B; supplementary online Fig. 4B) and compared with murine embryonic stem cells that have been stably transfected with the reverse transcriptase unit of telomerase and thus show high levels of telomerase activity (Armstrong et al., unpublished data, 2004). The telomerase activity has been shown to correlate to the cell cycle status of the cells . It is therefore tempting to suggest that the cycling cells present in the Lin–ALDHbright population have much higher levels of this enzyme than the cycling cells in the Lin– and Lin–ALDHlow population.
DISCUSSION
We would like to emphasize that marking of hematopoietic progenitors on the basis of ALDH activity using the new Aldefluor substrate provides an easy, reproducible, and convenient method for isolation of viable hematopoietic progenitors in clinical settings in which one aims to transplant long-term repopulating stem cells as well as transplant progenitor cells for short-term recovery after myeloablation therapy. In addition, it provides a powerful tool for isolating and comparing HSCs from different strains of mice for basic biology and aging studies. It will also prove useful for therapeutically directed studies that require isolation methods without the use of cell-surface antibodies for additional genetic manipulation such as nuclear transfer (in which the presence of antibodies may be harmful) from less terminally differentiated cells. Finally, it provides a powerful tool for isolating hematopoietic or other tissue-specific progenitors in cases in which their cell-surface phenotype is likely to be unknown such as from differentiation of embryonic stem cells.
The ability to obtain a population of cycling progenitor cells that can be used as targets for gene transfer and possession of relatively high levels of telomerase activity that facilitates the in vitro expansion of the cells before transplantation provides an excellent alternative to transduction strategies based on attempts of influencing the cell cycle of stem cells.
ACKNOWLEDGMENTS
Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661–673.
Osawa M, Hanada K, Hamada H et al. Long-term lympho-hematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94: 5320–5325.
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34–Lin– and CD34+Lin– hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 2000;95:2813–2820.
Srour EF, Brandt JE, Briddell RA et al. Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. Blood 1993;81:661–669.
Kim MJ, Cooper DC, Hayes SF et al. Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 1998;91:4106–4117.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Li CL, Johnson GR. Rhodamine123 reveals heterogeneity within murine Lin–, Sca-1+ hemopoietic stem cells. J Exp Med 1992;175:1443–1447.
Russo JE, Hilton J. Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells. Cancer Res 1988;48:2963.
Labrecque J, Bhat PJ, Lacroix A et al. Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidises retinol to retinoic acid. Biochem Cell Biol 1993;71:85.
Russo JE, Hilton J, Colvin OM. The role of aldehyde dehydrogenase isozymes in cellular resistance to the alkylating agent cyclophosphamide. In: Weiner H, Flynn TG, eds. Enzymology and Molecular Biology of Carbonyl Metabolism 2. New York: Liss, 1989:65.
Jones RJ, Barber JP, Vala MS et al. Assesment of aldehyde dehydrogenase in viable cells. Blood 1995;85:2742–2746.
Jones RJ, Collector MI, Barber JP et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen-colony forming activity. Blood 1996;88:487–491.
Storms RW, Trujillo AP, Springer JB et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci U S A 1999;96:9118–9123.
Hess DA, Meyerrose TR, Wirthlin L et al. Functional characterization of highly purified human hematopoietic repopulating cells isolated based on aldehyde dehydrogenase activity. Blood 10.1182/blood-2004-02-0448.
Ogawa M. Changing phenotypes of haematopoietic stem cells. Exp Hematol 2002; 30:3.
Drew E, Merkens H, Chelliah S et al. CD34 is a specific marker of murine mast cells. Exp Hematol 2002;30:1211–1218.
Higuchi Y, Zaeng H, Ogawa M. CD38 expression by hematopoietic stem cells of newborn and juvenile mice. Leukemia 2003;17:171–174.
Nilsson SK, Haylock DN, Johnston HM et al. Hyaluronan is synthesized by primitive hematopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differentiation in vitro. Blood 2003;101:856–862.
Avigdor A, Goichberg P, Shivtiel S et al. CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to the bone marrow. Blood 2004;103:2981–2989.
Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 1993;82:3327–3332.
Christinsen JL, Weissman IL. Flk-2 is a marker in hema-topoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001; 98:14541–14546.
Notaro R, Cimmino A, Tabarini D et al. In vivo telomere dynamics of human hematopoetic stem cells. Proc Natl Acad Sci U S A 1997;94:13782–13785.
Armstrong L, Lako M, von Herpe I et al. A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonal stem cell differentiation. Mech Dev 121; 12: 1509–1522.
b BD Biosciences, Oxford, United Kingdom;
c Department of Biological Sciences, University of Durham, Durham, United Kingdom
Key Words. Hematopoietic stem cells ? Aldehyde dehydrogenase activity ? Telomerase activity ? Murine hematopoietic progenitor cells
Correspondence: Majlinda Lako, Ph.D., Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00-44-191-241-8688; Fax: 00-44-191-241-8666; e-mail: Majlinda.Lako@ncl.ac.uk
ABSTRACT
Hematopoietic stem cells (HSCs) are multipotent cells that are normally resident in the bone marrow and are responsible for producing all of the adult hematopoietic lineages. They can be enriched from bone marrow and used for transplantation purposes, and the ease of accessibility of the bone marrow makes them an attractive model system in which to investigate the stem cell biology of renewing tissues. Most enrichment procedures for HSCs have involved the fractionation of cells based on the expression of cell-surface antigens or functional characteristics of the cells such as low-intensity fluorescence of Rhodamine 123 or Hoechst 33342 staining . There are several problems associated with these techniques. Primarily, the isolated populations, although highly enriched, still contain significant numbers of more mature progenitor cells . Second, the expression of surface antigens has been shown to vary among species, genetic background, and stem cell source . Some of these techniques require excitation at 350 nm (UV range), which may be harmful to the target cells. These procedures can be technically difficult and do not readily lend themselves to clinical applications. It is desirable, therefore, to develop alternative methods by which stem cell isolation may be achieved.
The aldehyde dehydrogenases (ALDHs) are intracellular enzymes responsible for oxidizing aldehydes to carboxylic acids . Members of this class are central to the processing of ethanol and amines produced during the catabolism of catecholamines and the conversion of vitamin A to retinoic acid . Both the hematopoietic progenitor cells and intestinal crypt cells display high levels of cytosolic ALDH and are relatively resistant to cyclophosphamide . To make use of this feature for purification of HSCs, several groups have developed fluorescent substrates for these enzymes. The first of these was based on the oxidation of dansylaminoacetaldehyde to dansylglycine . Jones et al. used this substrate to purify viable hematopoietic progenitors that were able to confer delayed but stable engraftment of myeloablated recipients and lacked characteristic cell-surface HSC markers. This substrate suffers from the disadvantage of UV excitation, which could be mutagenic to the cells, and the emission spectrum of this substrate overlaps with other fluorochromes and cannot readily be combined with other stem cell markers. In 1996, Storms et al. devised a new system that uses a visible light excitable fluorophore (BODIPY-conjugated aminoacetaldehyde) that is metabolized by ALDH to a carboxylate ion retained within the cell, allowing cells with high levels of ALDH to be isolated by fluorescence-activated cell sorter (FACS) because of their high fluorescence. Using a two-step strategy that combines the depletion of cells that express mature lineage markers with high activity of ALDH, highly purified hematopoietic-repopulating cells were purified from human umbilical cord blood using the BODIPY-based substrate named Aldefluor. This substrate had not been applied to murine models, and we wished therefore to investigate its application for the purification of murine hematopoietic progenitor from bone marrow. Using lineage depletion followed by Aldefluor staining, we were able to isolate a highly purified population of hematopoietic progenitors from murine bone marrow of both C57BL6 and BALB/c mice. Furthermore, we have shown that Lin–ALDHbright cells show cell-surface marker characteristics of primitive hematopoietic progenitors and are highly quiescent and enriched for hematopoietic progenitor activity.
MATERIALS AND METHODS
Purification of Lin–ALDHbright and Lin–ALDHlow Cells
To enrich for hematopoietic progenitors, we carried out a lineage depletion of committed cells using anti-PE magnetic beads (Miltenyi Biotech) and antibodies directed against lymphoid, myeloid, and erythroid cells. The N,N-diethyl-aminobenzaldehyde inhibitor sample was analyzed first using FL1 and side scatter suggested by the manufacturer, and the R1 gate was chosen to represent the background level of cytosolic ALDH activity (Lin–ALDHlow; Fig. 1A). Staining of Lin– cells with Aldefluor but without the inhibitor caused a shift in fluorescence, which allowed us to define the Lin–ALDHbright cells in gate R2 (Fig. 1B). The R2 region comprised 26.8 ± 1.0% of the Lin– population in C57BL6 mice and 0.058 ± 0.01% of the total bone marrow cells (data derived from seven independent experiments). Similarly, in BALB/c mice, the R2 region contained 23.5 ± 1.0% of the Lin– population and 0.055 ± 0.01% of the total population (data derived from three independent experiments; supplementary online Fig. 1B).
Figure 1. Purification of Lin–ALDHbright and Lin–ALDHlow cell populations in C57BL6 mice. Murine bone marrow cells were subjected to lineage depletion before staining with Aldefluor substrate. (A): Lin– cells stained with Aldefluor substrate and DEAB inhibitor were analyzed first to establish the baseline level of ALDH activity (gate R1, Lin–ALDHlow cell population); a dot plot and analysis contour plot are shown on the left and right sides, respectively. (B): Staining of Lin– cells with Aldefluor but without DEAB inhibitor produced a shift in fluorescence that defined the Lin–ALDHbright population (R2 gate; 26.8 ± 1.0% of the Lin– population); a dot plot and analysis contour plot are shown on the left and right sides, respectively. The data represent the mean ± standard error of the mean performed on seven independent Lin– cells purified from murine bone marrow. Abbreviations: ALDH, aldehyde dehydrogenase; DEAB, N,N-diethylaminobenzaldehyde.
Characterization of Lin–ALDHbright and Lin–ALDHlow Cells
Phenotypic investigation of Lin–ALDHbright cells in C57BL6 mice was performed using cell-surface markers common to the hematopoietic lineage (CD45; Figs. 2A, 2B), hematopoietic stem cell markers (c-kit, CD34, Sca-1, Flk-2, CD38, CD44; Figs. 2C–2J, 2M–2P), endothelial progenitor marker, Flk-1 (Figs. 2K, 2L), and mesenchymal stem cell markers (CD13, CD81, CD29; Figs. 2Q–2V). The Lin–ALDHbright population was comprised entirely of hematopoietic cells and devoid of mesenchymal or endothelial progenitors, as shown by specific marker staining. In addition, the Lin–ALDHbright population contained a higher percentage of cells that expressed the HSC marker c-kit compared with Lin–ALDHlow cells (Figs. 2C, 2D) but lacked expression of Sca-1 (Figs. 2G, 2H). Lin–ALDHbright cells were mostly devoid of CD34 (Figs. 2I, 2J) and CD38 expression (Figs. 2M, 2N), as demonstrated previously for murine hematopoietic stem cells in newborn and juvenile mice . Higuchi et al. have reported that a CD38+ subpopulation of HSCs appears before the age of 5 weeks and expands during adolescence. To ensure that we did not miss the developmental time point at which CD38 expression is observed in murine HSCs, we repeated the CD38 phenotypic analysis of Lin–ALDHbright at weeks 12, 14, and 16 (data not shown). We noticed no change in CD38 expression in the Lin–ALDHbright population between 8–12 weeks of development and 16 weeks, suggesting no expansion in CD38+ progenitors in the Lin–ALDHbright during this interval. Because CD38 expression is noticeable upon activation of juvenile HSCs in mice, it is tempting to speculate that the hematopoietic progenitors marked by high levels of ALDH activity are quiescent and might gain CD38 expression upon activation, an investigation that is now underway in our laboratory.
Figure 2. Phenotypic characterization of Lin–ALDHbright and Lin–ALDHlow cell populations in C57BL6 mice. Lin–ALDHbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and analyzed for the expression of CD45 (A, B), c-kit (C, D), Flk-2 (E, F), Sca-1 (G, H), CD34 (I, J), Flk-1 (K, L), CD38 (M, N), CD44 (O, P), CD29 (Q, R), CD81 (S, T), and CD13 (U, V). Data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. Statistical significance of the change between Lin–ALDHbright and Lin–ALDHlow cell populations was calculated using Student’s t-test and is shown as p value for each antibody staining.
All Lin–ALDHbright cells expressed CD44 (Figs. 2O, 2P), a receptor for the cell-surface adhesion molecule hyaluronic acid, which has been shown to be synthesized by primitive hematopoietic cells and is involved in their lodgment to the bone marrow and their proliferation . The classical definition of HSC phenotype in mice includes low expression of Thy-1.1 and high expression of Sca-1; however, this is strain dependent and applicable only to mice expressing the Ly-6A/E and Thy-1.1 haplotypes . C57BL6/J mice express the Thy-1.2 haplotype, and although it has been shown that Thy1.2+ c-kit+ Sca-1+ Lin–/low cells are HSCs, the level of Thy1.2 expression is lower than Thy-1.1, causing Thy1.2+ cells to bleed into the negative gate. In view of these results, it was suggested recently that HSCs from Thy1.2 strains can be isolated by adding Flk-2 to the lineage cocktail . In addition, the loss of Thy-1.1 expression and gain of Flk-2 has been correlated to loss of self-renewal during HSC maturation, such that HSCs lacking Flk-2 have long-term repopulation ability, whereas HSCs with Flk-2 expression show short-term repopulating capacity . Our study showed that Lin–ALDHbright has fewer Flk-2+ cells compared with Lin–ALDHlow (Figs. 2E, 2F), suggesting that although separation on the basis of ALDH activity enriches for primitive progenitor cells, it is likely to include cells with both short- and long-term repopulating ability.
We repeated the same phenotypic analysis on BALB/c mice, which are of different genetic background to C57BL6 mice. We found that Lin–ALDHbright cells were comprised entirely of hematopoietic cells and mostly devoid of mesenchymal or endothelial cells (supplementary online Figs. 2A, 2B, 2M, 2N, 2T–2Y), as were Lin–ALDHbright cells in C57BL6 mice. Lin–ALDHbright cells showed expression of typical HSC markers, such as c-kit and CD44 (supplementary online Figs. 2C–2D, 2R–2S), and mostly lacked CD34 and CD38 expression (supplementary online Figs. 2K, 2L, 2P, 2Q). We did find very low expression of Sca-1 in both Lin–ALDHbright and Lin–ALDHlow cells (supplementary online Figs. 2I, 2J), and this is consistent with previous findings reported by Spangrude and Brooks for the Lin– of BALB/c mice. We found a small percentage of Flk-2+ cells in the Lin–ALDHbright cells (supplementary online Figs. 2E, 2F), suggesting the presence of short-term repopulating cells in the Lin–ALDHbright cells. This analysis showed us that despite genetic background, Lin–ALDHbright cells show phenotypic similarities for the main HSC markers such as c-kit, CD34, CD38, and CD44.
Characterization of Lin–ALDHbright and Lin–ALDHlow Using In Vitro Colony Assays
Cell-surface staining predicted that the Lin–ALDHbright population is likely to be enriched for primitive hematopoietic progenitors. To investigate this possibility, we carried out in vitro colony-forming assays, which allowed us to estimate the plating efficiency of the cells and the nature of progenitors (Fig. 3, Table 1; supplementary online Figs. 3A, 3B). Comparison of the Lin–ALDHbright and Lin–ALDHlow populations showed that the Lin–ALDHbright contained all of the hematopoietic progenitor activity of Lin– cells in both C57BL6 and BABL/C mice, as demonstrated by colony assays (Fig. 3; supplementary online Fig. 3A) and an increased plating efficiency compared with Lin–ALDHlow and Lin– cells (Table 1; supplementary online Fig. 3B). Recent findings have also shown that the Lin–ALDHbright population of umbilical cord blood contains all of the hematopoietic progenitor activity and provides a better tool for resolving the functional differences in repopulating ability between human CD34+ cells . Despite the presence of c-kit expression, Lin–ALDHlow cells did not show any hematopoietic progenitor activity, which indicates that there are functional differences within the c-kit population that can be resolved on the basis of ALDH activity.
Figure 3. Lin–ALDHbright cell population of C57BL6 mice is enriched for hematopoietic progenitor activity. Lin–ALDHbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and subsequently sorted on a BD FACSVantage. In vitro colony assays were performed with Lin–, Lin–ALDHbright, and Lin–ALDHlow cell populations to define the progenitor activity and the plating efficiency of these cells. Data represent the number of individual colonies produced per 100 cells and represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow.
Table 1. Number of colonies per 100 cells
Cell Cycle Status of Lin–ALDHbright and Lin–ALDHlow Cells
Various studies have shown that murine HSCs enter the cell cycle less frequently . We performed cell cycle analysis on Lin–ALDHbright and Lin–ALDHlow cells of C57BL6 mice and found that the Lin–ALDHbright has the lowest content of cycling cells, averaging 10.8 ± 0.7% (Fig. 4A) compared with the Lin–ALDHlow population, which contained 41.5 ± 1.0% of cells in S phase (p = .002, n = 3). Similar results were obtained with the Lin–ALDHbright and Lin–ALDHlow cells of BALB/c mice (supplementary online Fig. 4A). This is consistent with the prediction that these cells contain more primitive progenitors that are quiescent or at least do not contribute to hematopoiesis at any given time .
Figure 4. Cell cycle analysis and telomerase activity of Lin–ALDHbright and Lin–ALDHlow populations of C57BL6 mice. (A): Lin–ALD-Hbright and Lin–ALDHlow cell populations were defined as described in Figure 1 and subsequently sorted on a BD FACSVantage. Phosphatidylinositol staining was used to define the DNA content of the two populations. The data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. (B): Telomerase activity of Lin–ALDHbright and Lin–ALDHlow cell populations was measured using telomeric repeat amplification protocol assays and 1 ng of protein extract as described in Materials and Methods. Data represent the mean ± standard error of the mean performed on three independent Lin– cells purified from murine bone marrow. Statistical significance of the change between Lin–ALDHbright and Lin–ALDHlow cell populations was calculated using Student’s t-test.
Lin–ALDHbright Cells Have Higher Telomerase Activity Than Lin–ALDHlow Cells
Most cycling HSCs display telomerase activity, and this decreases as HSCs proliferate and differentiate into more mature cells that display negligible levels of this enzyme . To measure telomerase activity, we performed TRAP assays under linear conditions of product amplification using a range of dilutions of protein extracts that we have previously demonstrated to be a suitable range for cells with relatively high telomerase activity . We found that Lin–ALDHbright cells of both C57BL6 and BALB/c mice have significantly higher levels of telomerase compared with Lin–cells and Lin–ALDHlow (Fig. 4B; supplementary online Fig. 4B) and compared with murine embryonic stem cells that have been stably transfected with the reverse transcriptase unit of telomerase and thus show high levels of telomerase activity (Armstrong et al., unpublished data, 2004). The telomerase activity has been shown to correlate to the cell cycle status of the cells . It is therefore tempting to suggest that the cycling cells present in the Lin–ALDHbright population have much higher levels of this enzyme than the cycling cells in the Lin– and Lin–ALDHlow population.
DISCUSSION
We would like to emphasize that marking of hematopoietic progenitors on the basis of ALDH activity using the new Aldefluor substrate provides an easy, reproducible, and convenient method for isolation of viable hematopoietic progenitors in clinical settings in which one aims to transplant long-term repopulating stem cells as well as transplant progenitor cells for short-term recovery after myeloablation therapy. In addition, it provides a powerful tool for isolating and comparing HSCs from different strains of mice for basic biology and aging studies. It will also prove useful for therapeutically directed studies that require isolation methods without the use of cell-surface antibodies for additional genetic manipulation such as nuclear transfer (in which the presence of antibodies may be harmful) from less terminally differentiated cells. Finally, it provides a powerful tool for isolating hematopoietic or other tissue-specific progenitors in cases in which their cell-surface phenotype is likely to be unknown such as from differentiation of embryonic stem cells.
The ability to obtain a population of cycling progenitor cells that can be used as targets for gene transfer and possession of relatively high levels of telomerase activity that facilitates the in vitro expansion of the cells before transplantation provides an excellent alternative to transduction strategies based on attempts of influencing the cell cycle of stem cells.
ACKNOWLEDGMENTS
Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661–673.
Osawa M, Hanada K, Hamada H et al. Long-term lympho-hematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
Bhatia M, Wang JC, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94: 5320–5325.
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34–Lin– and CD34+Lin– hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 2000;95:2813–2820.
Srour EF, Brandt JE, Briddell RA et al. Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro. Blood 1993;81:661–669.
Kim MJ, Cooper DC, Hayes SF et al. Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 1998;91:4106–4117.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Li CL, Johnson GR. Rhodamine123 reveals heterogeneity within murine Lin–, Sca-1+ hemopoietic stem cells. J Exp Med 1992;175:1443–1447.
Russo JE, Hilton J. Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells. Cancer Res 1988;48:2963.
Labrecque J, Bhat PJ, Lacroix A et al. Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidises retinol to retinoic acid. Biochem Cell Biol 1993;71:85.
Russo JE, Hilton J, Colvin OM. The role of aldehyde dehydrogenase isozymes in cellular resistance to the alkylating agent cyclophosphamide. In: Weiner H, Flynn TG, eds. Enzymology and Molecular Biology of Carbonyl Metabolism 2. New York: Liss, 1989:65.
Jones RJ, Barber JP, Vala MS et al. Assesment of aldehyde dehydrogenase in viable cells. Blood 1995;85:2742–2746.
Jones RJ, Collector MI, Barber JP et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen-colony forming activity. Blood 1996;88:487–491.
Storms RW, Trujillo AP, Springer JB et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci U S A 1999;96:9118–9123.
Hess DA, Meyerrose TR, Wirthlin L et al. Functional characterization of highly purified human hematopoietic repopulating cells isolated based on aldehyde dehydrogenase activity. Blood 10.1182/blood-2004-02-0448.
Ogawa M. Changing phenotypes of haematopoietic stem cells. Exp Hematol 2002; 30:3.
Drew E, Merkens H, Chelliah S et al. CD34 is a specific marker of murine mast cells. Exp Hematol 2002;30:1211–1218.
Higuchi Y, Zaeng H, Ogawa M. CD38 expression by hematopoietic stem cells of newborn and juvenile mice. Leukemia 2003;17:171–174.
Nilsson SK, Haylock DN, Johnston HM et al. Hyaluronan is synthesized by primitive hematopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differentiation in vitro. Blood 2003;101:856–862.
Avigdor A, Goichberg P, Shivtiel S et al. CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to the bone marrow. Blood 2004;103:2981–2989.
Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 1993;82:3327–3332.
Christinsen JL, Weissman IL. Flk-2 is a marker in hema-topoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001; 98:14541–14546.
Notaro R, Cimmino A, Tabarini D et al. In vivo telomere dynamics of human hematopoetic stem cells. Proc Natl Acad Sci U S A 1997;94:13782–13785.
Armstrong L, Lako M, von Herpe I et al. A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonal stem cell differentiation. Mech Dev 121; 12: 1509–1522.