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

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.HSCs, defined by their ability to self-renew and to differentiate into all blood cell types, form the basis of bone marrow transplantation for treatment of cancers and hematopoietic disorders 1 , and are also a promising cell target for gene therapies that can potentially treat a broad variety of human diseases 2 . Development of these important clinical applications of HSCs is greatly hampered by the lack of understanding of the extracellular and intracellular signals that govern their fates as well as by the difficulty in carrying out ex vivo expansion of these cells.Numerous attempts have been made to increase the number of long-term HSCs in culture 3,4 . The use of stromal cell lines or combinations of cytokines have resulted in considerable selfrenewal of mouse HSCs assayed 4-6 weeks after transplant, and have led to as much as a sixfold increase in mouse long-term HSC activity in culture [5][6][7][8][9] . The introduction of exogenous transcription factors can expand HSCs more substantially [10][11][12] 13 . We subsequently developed a simple culture system using low but saturating levels of stem cell factor (SCF), thrombopoietin (TPO), IGF-2 and fibroblast growth factor 1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a greater than eightfold increase in numbers of long-term repopulating HSCs (LT-HSCs) after 10 d of culture of highly enriched bone marrow HSCs 14 .Here we further analyzed gene expression of E15 mouse liver CD3 + cells using Affymetrix U74Bv2 and U74Cv2 mouse microarrays, and identified the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3) as well as several other members of the family of angiopoietin-like proteins as potent stimulators of ex vivo expansion of HSCs. RESULTS Fetal liver CD3 + cells specifically express Angptl2 and Angptl3In Supplementary Fig. 1 online). These proteins are also expressed in adult bone marrow cells, including the side population (SP) CD45 + Sca-1 + highly enriched HSC population 17 ( Supplementary Fig. 1 online). We therefore hypothesized that these two proteins, not previously thought to be involved in HSC biology, might support expansion of HSCs. Angptl2 and Angptl3 stimulate ex ...

Atypical angiopoietin-like protein that regulates ANGPTL3

“…T he angiopoietin-like (ANGPTL) genes encode a family of secreted proteins with pleiotropic effects on vascular cells (1), lipid metabolism (2), and stem cell biology (3). The family members share a common architecture, comprising an extended N-terminal domain and a C-terminal fibrinogen-like domain.…”

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

“…For example, the low absolute numbers of HSCs in most cord blood samples [3] [4] restrict the clinical utility of these products. A method for significantly expanding HSCs could not only address these insufficiencies but also broaden the exploitation of reduced conditioning regimens and associated therapeutic benefits.One approach that has allowed some HSC expansion in vitro to be achieved has focused on the identification of optimized combinations and concentrations of externally acting growth factors and related molecules [5][6][7][8][9][10][11][12]. A complementary approach has been to identify intrinsic regulators such as transcription factors [13] and key mediators of signaling pathways [14,15] that can be manipulated to activate or promote HSC self-renewal divisions.…”

Ex vivo expansion of hematopoietic stem cells

“…The abilities of many cytokines to support hematopoietic progenitors to form colonies in vitro provided important insights into expansion of functional primitive long-term (LT-) HSCs that are measured by in vivo repopulating activity [49]. In the last two decades, a number of secreted/extracellular proteins/chemicals have been demonstrated to support ex vivo expansion of mouse HSCs, including stem cell factor (SCF) [50], thrombopoietin (TPO) [51][52][53], Notch ligands [54,55], Wnt ligands [56][57][58][59], fibroblast growth factor 1 (FGF-1) [60,61], bone morphogenetic proteins (BMPs) [62], Hedgehogs [62][63][64], prostaglandin E2 (PGE2) [65], interleukin 10 (IL-10) [66], insulin-like growth factor 2 (IGF-2) [67,68], IGF binding protein 2 (IGFBP2) [69,70], several angiopoietin-like proteins (Angptls) [71][72][73][74], and pleiotrophin [75]. Conditional derivatives of certain growth factor receptors have also been used to support HSC expansion in culture [76,77].…”

“…In parallel, the knowledge gained from the co-culture of HSCs with various stromal cell types, including aorta-gonado-mesonephros (AGM), fetal liver, and bone marrow stromal cells, and with endothelial cells and cancer cells has provided important guidance for development of ex vivo expansion strategies in medium with defined factors [67,68,71,[81][82][83][84][85]. The Williams lab [83] established stromal cell lines from yolk sac that support the activities of HSCs and hematopoietic progenitors.…”