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Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells

Nature Medicine volume 12, pages 240–245 (2006)Cite this article

Abstract

Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3+ cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

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Figure 1: Angptl2 substantially stimulates ex vivo expansion of HSCs.
Figure 2: Immunoaffinity purification of Angptl2 from conditioned medium of transfected 293T cells.
Figure 3: Purified Angptl2 or Angptl3 stimulates ex vivo expansion of HSCs, measured by a limiting dilution assay.
Figure 4: Mammalian cell-specific post-translational modifications and the coiled-coil domain of Angptl2 are important for stimulation of ex vivo expansion of HSCs.
Figure 5: Purified mouse Angptl3, human Angptl5, human Angptl7 or human Mfap4 stimulated ex vivo expansion of HSCs.

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Acknowledgements

We thank R&D Systems Inc. for providing purified bacterially expressed Angptl2, insect-expressed Angptl3 and bacterially expressed Angptl7, as well as monoclonal antibodies against human Angptl2. We are grateful to B. Zhou for help in protein expression, G. Paradis and M. Jennings in Massachusetts Institute of Technology flow cytometry core facility for cell sorting, and A. Babic for critically reading the manuscript. C.C.Z. is a Leukemia and Lymphoma Society Fellow. H.F.L. was supported by US National Institutes of Health grant R01 DK 067356 and from the Engineering Research Centers Program of the National Science Foundation under National Science Foundation Award Number EEC 9843342 through the Biotechnology Process Engineering Center at the Massachusetts Institute of Technology.

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Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, 02142, Massachusetts, USA

    Cheng Cheng Zhang, Megan Kaba, Guangtao Ge, Kathleen Xie, Wei Tong, Christopher Hug & Harvey F Lodish

  2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Christopher Hug

  3. Division of Respiratory Diseases, Children's Hospital, 300 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Christopher Hug

  4. Harvard Medical School, 25 Shattuck Street, Boston, 02115, Massachusetts, USA

    Harvey F Lodish

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  1. Cheng Cheng Zhang
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Correspondence to Harvey F Lodish.

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Supplementary information

Supplementary Fig. 1

Analysis of Angptl2 mRNA expression in hematopoietic cells from mouse day 15 fetal liver or adult bone marrow cells by real-time PCR. (PDF 80 kb)

Supplementary Fig. 2

The majority of freshly isolated BM HSCs and all cultured BM HSCs bind to Angptl2. (PDF 51 kb)

Supplementary Table 1

List of transcripts encoding secreted and membrane proteins that are abundant in fetal liver CD3+ cells and low in adult splenic CD3+ cells and fetal liver Gr-1+ cells, identified from Affymetrix U74Bv2 and U74Cv2 mouse chips. (PDF 29 kb)

Supplementary Methods (PDF 0 kb)

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Zhang, C., Kaba, M., Ge, G. et al. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med 12, 240–245 (2006). https://doi.org/10.1038/nm1342

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