Leukemia (1998) 12, 371–381 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions – comparison to other cytokines and growth factors J Ratajczak, Q Zhang, E Pertusini, BS Wojczyk, MA Wasik and MZ Ratajczak Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The role of insulin (INS), and insulin-like growth factor-I (IGF- has been difficult to assess. The fact that EpO alone fails to I) in the regulation of human erythropoiesis is not completely stimulate BFU-E in serum-free conditions, but does do in understood. To address this issue we employed several comp- lementary strategies including: serum free cloning of CD34؉ serum containing cultures indicates that serum contains some cells, RT-PCR, FACS analysis, and mRNA perturbation with oli- crucial growth factors necessary for the BFU-E development. godeoxynucleotides (ODN). In a serum-free culture model, both In previous studies from our laboratory, we examined the ؉ INS and IGF-I enhanced survival of CD34 cells, but neither of role of IGF-I12 and KL9,11,13 in the regulation of early human these growth factors stimulated their proliferation. The influ- erythropoiesis. Both of these growth factors are considered to ence of INS and IGF-I on erythroid colony development was be crucial for the BFU-E growth.3,6,8,14 Unexpectedly, that dependent on a combination of growth factors used for stimul- + ating BFU-E growth. When BFU-E growth was optimally stimu- addition of IGF-I to cultures of the purified human CD34 cells lated with erythropoietin (EpO) ؉ kit ligand (KL) the large did not enhance formation of the hematopoietic colonies.12 erythroid colonies developed normally even in the absence of Similarly, downregulation of IGF-I R using antisense oligo- INS or IGF-I. However, the addition of both of these growth fac- deoxynucleotides was without any apparent effect on the tors slightly enhanced colony size. On the other hand, if eruthroid colonies were stimulated suboptimally with EpO + IL- growth of normal human hematopoietic progenitors. In con- 3 only, INS or IGF-I increased the number of small erythroid trast, antisense oligodeoxynucleotides against c-Kit R totally bursts by ෂ30%. Both INS and IGF-I activated signal transduc- inhibited the BFU-E colony formation, if CD34+ cells were co- tion in maturing human erythropoietic cells as determined by stimulated with EpO + KL alone.9,11,13 We concluded there- phosphorylation of the insulin receptor substrate-2 (IRS-2) pro- fore, that KL, but not IGF-I, is a critical regulator of human tein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34؉, c-kit-R؉ marrow cells, and erythroid colony formation. We concluded also, that EpO is 11 in cells isolated from BFU-E and CFU-GM colonies. Expression not able to support BFU-E colony growth if KL is not present of INS-R protein on these cells was subsequently confirmed by in the cultures. This last observation was recently confirmed cytofluorometry. In contrast, the receptor for insulin-like 1–3 .؉ in several elegant studies by the other investigators growth factor-I (IGF-IR) was not detected on CD34 cells, and Nevertheless, since our previous studies were performed in was first easily detectable on more differentiated cells derived 8 from day 6 BFU-E and CFU-GM colonies. We conclude that INS the presence of serum, which is known to contain INS, the and IGF-I may be survival factors for human CD34؉ cells, but effect of IGF-I on colony formation was uncertain. As it is well are not required during early erythropoiesis. In contrast, both known, INS, IGF-I and IGF-II show a high degree of the mol- growth factors may play some role at the final stages of ecular homology, and crossreact with their corresponding erythroid maturation. receptors. In addition, INS R and IGF-I R are structurally Keywords: erythropoiesis; antisense oligonucleotides; insulin; insulin-like growth factors; insulin receptor; insulin-like growth factor related heterodimers, which possess intrinsic tyrosine kinase I receptor activity, and after interaction with IRS-1 and IRS-2 ‘docking proteins’ activate appropriate signal transduction proteins. In contrast, the third receptor of this family, IGF-II R, is a single Introduction highly glycosylated transmembrane peptide, which does not possess tyrosine kinase activity. The ability of IGF-II R to The formation of red blood cells is regulated by various cyto- recruit signal transduction proteins is not well documented in kines and growth factors. Of these, erythropoietin (EpO) is the literature.15 absolutely required for survival and differentiation of the To learn more about the role of the insulin growth factor erythropoietic progenitors.1–3 Other growth factors, and cyto- family in regulating human erythropoiesis, we employed a kines such as insulin (INS), insulin-like growth factors (IGF-I, well-defined serum-free cloning system to examine the direct IGF-II), basic fibroblast growth factor (FGF-2), hepatocyte effect of INS and IGF-I on survival and cloning efficiency of growth factors (HGF), prolactin (PRL), interleukins (IL-3, IL- the CD34+ cells. We also studied the development of BFU-E 4, IL-6, IL-9, IL-11), granulocyte–monocyte stimulatory factor colonies compared to the effect of other cytokines and growth (GM-CSF), and kit ligand (KL), have been reported to co-stimu- factors which have been reported to enhance erythropo- late with EpO the growth of the erythroid colonies.4–11 Since iesis.4,6,16–19 Finally, we analyzed by FACS and RT-PCR the several of these factors including KL, INS and IGF-I, are usu- expression of INS-R and IGF-IR in the early progenitor cells ally present in the serum used to supplement cell culture and their more differentiated, cultured derivatives. Our results media, contribution of the individual factors to erythropoiesis suggest that neither INS nor IGF-I are critical for human in experiments performed in serum supplemented conditions erythropoiesis. These factors seem to inhibit apoptosis in the human early hematopoietic cells but do not appear to affect the initial proliferation and differentiation of these cells. INS Correspondence: MZ Ratajczak, Rm 515 Stellar-Chance Laboratories, and IGF-I may, however contribute more effectively to the University of Pennsylvania School of Medicine, 422 Curie Blvd, Phila- final maturation of erythroid colonies. delphia, PA 19104, USA; Fax 215 573 2078 Received 23 May 1997; accepted 14 November 1997 Insulin and human hematopoiesis MZ Ratajczak et al 372 Materials and methods growth factor, 20 ng/ml), G-CSF (10 ng/ml), GM-CSF (5 ng/ml), IL-3 (20 U/ml) and KL (100 ng/ml). Recombinant Oligodeoxynucleotides (ODN) human growth factors were used in all experiments (R&D, Minneapolis, MN, USA). Colonies were counted with an Unmodified 18-base ODN were synthesized on an Applied inverted microscope on day 11 (CFU-GM), on day 14 (BFU- Biosystems (Foster City, CA, USA) 380B DNA synthesizer by E), and on day 15 (CFU-Mix). means of ␤-cyanoethyl phosphoroamidite chemistry as In some experiments cells were cultured in serum-free con- described. ODN were purified by ethanol precipitation and ditions with or without exogenously added insulin (20 ␮g/ml) multiple washes in 70% ethanol, lyophilized to dryness, and for 7 days before growth factors were added to stimulate re-dissolved in culture medium at a concentration of 1 mg/ml growth of CFU-Mix, BFU-E and CFU-GM colonies. (0.167 mmol/l). ODN sequences utilized were based on the published human IGF-I R sequence,20 and were targeted to codons −21−26 of the pro-receptor signal peptide: antisense Cell selection and mRNA isolation ODN; 5′-TCC TCC GGA GCC AGA CTT-3′, sense ODN: 5′- AAG TCT GGC TCC GGA GGA-3′ and scrambled ODN; 5′- A-T-MNC were enriched for CD34+ cells by immunoaffinity TGA GAC TCC TTA CCG CCG-3′ as described.21 selection with murine monoclonal antibody HPCA-1 (Becton Dickinson, San Jose, CA, USA) and magnetic beads according to the manufacturer’s protocol (Dynal, Oslo, Norway). The c- Cells kit R+ subset of CD34+ cells was isolated by FACS. In brief, 2 × 107 human A-T-MNC were suspended in PBS sup- Light-density marrow mononuclear cells (MNC) were plemented with 5% bovine calf serum (Hyclone, Logan, UT, obtained from 11 consenting healthy donors and depleted USA) (BCS) and labeled for 30 min at 4°C with anti-c-kit R of adherent cells and T lymphocytes (A-T-MNC) as monoclonal antibodies (SR-1) (1:1000) (generously provided described.10–12,22 by V Broudy, University of Washington, Seattle, WA, USA). Cells were washed three times in ice-cold PBS supplemented with 5% BCS and then incubated with phycoerythrin (PE)-con- Cell lines jugated goat anti-mouse monoclonal antibody (Sigma) (1:100) for 30 min at 4°C. Then the cells were washed again and incu- Chinese-hamster ovary (CHO) cell line transfected with bated with anti-CD34 monoclonal antibody conjugated with human INS-R was made available to us by P Rothenberg from fluorescein FITC anti-HPCA-2 (Becton Dickinson) (20 ␮l/106 the University of Pennsylvania. HTLV-1 positive T cell lines: cells) for 30 min at 4°C. After incubation, cells were washed HUT-102B and C91PL were obtained from G Trinchieri from three times in ice-cold PBS supplemented with 5% BCS and Wistar Institute, Philadelphia, and M Kamoun from the Uni- then subjected to fluorescence-activated cell sorting using a versity of Pennsylvania, respectively.