Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation

Cheng Cheng Zhang,Megan Kaba,Satoru Iizuka,Hoangdinh Huynh,Harvey F Lodish

doi:10.1182/blood-2007-11-122119

Abstract

AbstractHematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)–repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.

Highlights

Introduction bone marrow Hematopoietic stem cells (HSCs) in cultureHere, we measure the activity of multipotent human severe combined immunodeficient (SCID)–repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs
We showed that culture of mouse bone marrow side population (SP) CD45ϩSca-1ϩ cells in serum-free medium containing 10 ng/mL SCF, 20 ng/mL TPO, 20 ng/mL IGF-2, and 10 ng/mL FGF-1 (STIF medium) supplemented with one of several angiopoietin-like proteins (Angptls)— Angptl[2], Angptl[3], Angptl[5], Angptl[7], and Mfap4—stimulated a dramatic expansion of long-term repopulating HSCs (LT-HSCs) numbers and activities.[14]
We sought to test whether this culture could expand human cord blood HSCs

Summary

Introduction

Introduction bone marrow HSCs in cultureHere, we measure the activity of multipotent human severe combined immunodeficient (SCID)–repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation. Summers et al reported a very high incidence of long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) expansion in CD133ϩ cell cultures.[8] Araki et al demonstrated that the treatment of cord blood CD34ϩ cells with chromatin-modifying agents 5-aza-2Јdeoxycytidine and trichostatin A resulted in a 9.6-fold expansion of SRC numbers following 9 days of culture.[9] Using serum-free medium supplemented with SCF, TPO, Flt3-L, IL-3, IL-6/sIL-6R, and Delta 1, Suzuki et al reported an approximate 6-fold increase of SRC numbers.[10] In addition, a cell-permeable fusion protein containing the HOX B4 transcription factor, when expressed by stromal cells, stimulated ex vivo expansion of human SRCs.[11] So far, the proper mixture of growth factors and cytokines used in culture conditions has not yet been determined, and new growth factors for HSCs are needed

Methods

Results

Conclusion