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Comparative Study
. 1999 Apr 5;189(7):1139-48.
doi: 10.1084/jem.189.7.1139.

Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells

M Bhatia  1 , D Bonnet, D Wu, B Murdoch, J Wrana, L Gallacher, J E Dick
Affiliations
Comparative Study

Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells

M Bhatia et al. J Exp Med. .

Abstract

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34(+)CD38(-)Lin- stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor beta, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34(+)CD38(-) phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34(+) CD38(-)Lin- cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.

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Figures

Figure 1
Figure 1
Expression of BMP receptors and SMADs in primitive hematopoietic tissue derived from human sources. RT-PCR reactions were performed on human CD34+CD38−Lin− cells from CB (n = 4), BM (n = 2), M-PB (n = 2), and stromal cells (n = 2) as indicated for (A) BMP receptors ALK-3 and -6, and (B) human SMAD-1, -4, and -5. RT-PCR was performed on whole human fetus sample as a positive control for the reaction.
Figure 2
Figure 2
Effect of ex vivo culture on the total cell number and number of clonogenic progenitors present after in vitro culture of CD34+CD38−Lin− cells in the presence of BMPs. (A) Purified CD34+CD38−Lin− cells were counted and seeded (700–1,000) in wells containing serum-free media or with the addition of factors indicated at day 0. Cells were harvested from individual wells after 3 d of culture and counted, and the mean fold increase in absolute cell number was calculated (n = 4). (B) An aliquot of 100–300 CD34+CD38−Lin− cells was plated in progenitor cell assays at the initiation of ex vivo cultures (day 0), and the frequency of progenitors was calculated. Similar cell doses were plated from wells harvested after 3 d of cultures containing the various factors indicated, and the mean fold increase in number of CFCs was calculated compared with day 0 (n = 3). Values are the mean ± SEM of determinations in four and three separate culture samples for cell number and clonogenic progenitors, respectively. *P < 0.05, **P < 0.01 indicate statistically significant differences from controls.
Figure 3
Figure 3
Comparative analysis of CD34 and CD38 expression of highly purified CD34+CD38−Lin− cells after 4 d of culture in the presence of BMPs. A representative experiment (n = 3) of CD34 and CD38 cell surface expression performed on initially purified CD34+CD38−Lin− cells after 4 d of culture in serum-free conditions or with the addition of factors as indicated. The entire contents of individual wells were collected at 4 d, stained with mAbs, and analyzed using flow cytometric analysis.
Figure 3
Figure 3
Comparative analysis of CD34 and CD38 expression of highly purified CD34+CD38−Lin− cells after 4 d of culture in the presence of BMPs. A representative experiment (n = 3) of CD34 and CD38 cell surface expression performed on initially purified CD34+CD38−Lin− cells after 4 d of culture in serum-free conditions or with the addition of factors as indicated. The entire contents of individual wells were collected at 4 d, stained with mAbs, and analyzed using flow cytometric analysis.
Figure 4
Figure 4
Analysis of CD34 and CD38 expression of highly purified CD34+ CD38−Lin− cells after 6 d of culture in the presence of BMP-4. A representative experiment (n = 3) of CD34 and CD38 cell surface expression performed on initially purified CD34+CD38−Lin− cells after 6 d of culture in serum-free conditions or with the addition of BMP-4 at 5 or 25 ng/ml. The entire contents of individual wells were collected at 6 d, stained with mAbs, and analyzed using flow cytometric analysis.
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