And Insulin-Like Growth Factor-I (IGF-I) in Regulating Human Erythropoiesis

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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 complementary 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. The EBV-transformed FACS Star Plus II (Becton Dickinson). mRNA was extracted lymphoblastoid B cell line (LCL) was described previously.23,24 from the sorted population for RT-PCR analysis of gene expression using QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, Millwaukee, WI, USA), according to the Cell cultures manufacturer’s protocol.

Briefly, (104) CD34+ A-T-BMNC cells were cloned in 1 ml of medium containing 0.8% methylcellulose (Methocel MC, FACS analysis of marrow MNC Fluka, Switzerland) in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco BRL, Grand Island, NY, USA) supplemented Briefly, 1 × 106 human A-T-MNC were suspended in PBS sup- with 1% delipidated, deionized, and charcoal stripped bovine plemented with 5% BCS and labeled for 30 min at 4°C with serum albumine (BSA) (Sigma, St Louis, MO, USA), or albumin anti-INS-R antibody directed against epitope 83-14 of a sub- bovine RIA grade (INS Ͻ0.1 mU/mg) (Sigma), 270 ␮g/ml iron- unit of INS-R (the kind gift of Dr P Rothenberg, University saturated transferrin (Sigma), 5.6 ␮g/ml cholesterol (Sigma) of Pennsylvania, Philadelphia, PA) (1:2000)25 or anti-IGF-1R and 2 mmol/l l-glutamine. The appropriate growth factors antibody directed to the extracellular parts of alpha and beta were added to the mixture which was then transferred to 3.5- subunits of IGF-IR (Oncogene Science, Cambridge, MA, USA) ° cm plastic petri dishes and incubated (37 C, 95% air, 5% CO2 (1:100). Cells were washed three times in ice-cold PBS sup- humidified atmosphere) for the times appropriate to the colon- plemented with 5% BCS and then incubated with PE-conju- ies being grown. Exogenous growth factors were employed gated goat anti-mouse monoclonal antibody (Sigma) (1:100) as follows: erythropoietin (Epo; 5 U/ml) + interleukin-3 (IL-3; for 30 min at 4°C. The cells were washed and incubated with 20 U/ml), or EpO + kit ligand (KL; 100 ng/ml) for BFU-E, IL-3 FITC-HPCA-2 monoclonal antibody (Becton Dickinson) (20 U/ml) + granulocyte–macrophage colony-stimulating fac- (20 ␮l/106 cells) for 30 min at 4°C. After incubation, cells were tor (GM-CSF; 5 ng/ml) for CFU-GM, Epo (5 U/ml) + IL-3 again washed three times in ice-cold PBS supplemented with (20 U/ml) + GM-CSF (5 ng/ml) + KL (100 ng/ml) for CFU-Mix. 5% BCS and subjected to analysis using a FACS Star Plus II In some experiments BFU-E growth was co-stimulated with (Becton Dickinson). the optimal doses of following factors: INS (5–10 ␮g/ml), IGF- Specificity of the anti-INS-R and anti-IGF-IR antibodies was I (50 ng/ml), IGF-II (50 ng/ml), FGF-1 (50 ng/ml), FGF-2 confirmed by analysis of the cell lines infected with HTLV-I or (100 ng/ml), leukemia inhibitory factor (LIF, 50 ng/ml), IL-1␣ EBV. HTLV-I-positive cell lines have been shown previously to (100 pg/ml), IL-1␤ (100 pg/ml), IL-4 (50 ng/ml), IL-6 (80 U/ml), express high concentration of IGF-IR in ligand-binding assays. IL-9 (100 ng/ml), IL-11 (100 U/ml), VEGF (vascular endothelial We confirmed this high expression using the anti-IGF-IR anti- growth factor, 20 ng/ml), PRL (20 ng/ml), STK-1L (stem cell body. Furthermore, by also using the anti-INS-R antibody, we tyrosine kinase-1 receptor ligand, 20 ng/ml), HGF (hepatocyte have found that one of the HTLV-I+ lines, HUT-102B, Insulin and human hematopoiesis MZ Ratajczak et al 373 expresses only IGF-IR, whereas another, C91PL, expresses cipitate the immune complexes. The immunoprecipitates both receptors. An EBV-positive LCL line displays only the were separated on a 10% polyacrylamide-SDS gel, and trans- INS-R but not the IGF-IR (data not presented). ferred electrophoretically onto hybridization membranes. The membranes, were incubated with the murine 4G10 anti-phosphotyrosine monoclonal antibody (UBI, Lake Placid, NY, FACS analysis of cultured cells for INS-R and IGF-IR USA) followed by the peroxidase-conjugated anti-mouse Ab expression (Jackson Immuno-Research, West Grove, PA, USA). Blots were developed using the ECL chemiluminescence reagents Cells from day 6 or day 9 BFU-E and CFU-GM colonies were (Amersham Life Science, Arlington Heights, IL, USA). Auto- isolated from serum-free methylcellulose cultures by aspir- radiographs were quantitatively analyzed using a Molecular ation with a Pasteur pipette, washed twice in ice-cold PBS Dynamics Densitometer and Imagequant Version 3.22 supplemented with 5% BCS before incubation with anti-INS- software (Molecular Dynamics, Sunnyville, CA, USA). R Ab (1:2000) or IGF-1R Ab (1:100), followed by PE-conjugated goat anti-mouse monoclonal antibody (Sigma) (1:100) for 30 min at 4°C. After incubation, cells were again washed Isolation of mRNA from BFU-E and CFU-GM three times in ice-cold PBS supplemented with 5% BCS and subjected to analysis using a FACS Star Plus II (Becton BFU-E and CFU-GM colonies grown in serum-free methylcel- Dickinson). lulose cultures were analyzed for INS-R and IGF-IR expression by RT-PCR. Briefly, colonies were picked from day 6 to day 9 cultures using a Pasteur pipette under an inverted microscope. Detection of apoptosis Randomly selected colonies (ෂ20) from each culture dish were picked for each mRNA extraction. Colonies were resus- CD34+, Kit-R+ MNC (105) were isolated by FACS and then pended in 10 ml of IMDM and incubated for 1 h at 37°Cto resuspended in Iscove DMEM containing the serum-free sup- dissolve the methylcellulose, centrifuged, washed twice in plement described above. The cells were cultured in 6-well PBS, and mRNA was extracted. In brief, using QuickPrep plastic plates in the presence or absence of INS (20 ␮g/ml) for Micro mRNA Purification Kit (Pharmacia Biotech), according ° 72 h (37 C, 95% humidity, 5% CO2) and then washed once to the manufacturer’s protocol cells from 20 colonies were in PBS and fixed in 4% neutral-buffered formalin. After fix- lysed in 200 ␮l of RNAzol (Cinna-Biotecx, Houston, TX, ation, the cells were sedimented by gravity onto slides USA) + 22 ␮l of chloroform. The aqueous phase was collected covered with Cell-Tak tissue adhesive (Collaborative Biomed- and mixed with 1 volume of isopropanol (Sigma). RNA was ical Products, Bedford, MA, USA). Apoptosis was detected in precipitated overnight at −20°C. The RNA pellet was washed these cells using in situ apoptosis detection kit: ApopTag in 75% ethanol and resuspended in three times autoclaved

(Oncor, Gaithersburg, MD, USA) according to the manufac- H2O. turer’s protocol. In this assay, terminal deoxynucleotide trans- ferase (TdT) is used to label the fragmented ends of DNA which has undergone apoptosis with digoxigenin-dUTP. The Reverse transcriptase-polymerase chain reaction (RT- digoxigenin label is subsequently detected with a fluor- PCR) escently labeled anti-digoxigenin antibody allowing simple, direct enumeration of apoptotic cells with a fluorescence RT-PCR was carried out as described.10–12,22 Briefly, mRNA microscope. (0.5 ␮g) was reverse-transcribed with 500 U of Moloney murine leukemia virus reverse transcriptase (MoMLV-RT) and 50 pmol of an ODN primer complementary to the 3′ end of ODN exposure and cell cultures the following sequence of IGF-IR (nts 1641–1659): ATA CTC TGT GAC ATT CTT AA or INS R (nts 881–859): Cells were exposed to ODN as described.10–12,22 Briefly, CAG TCC TGG AAG TGG TAG TAC. The resulting cDNA CD34+ MNC (2 × 104) were incubated in 0.4 ml of IMDM sup- fragments were amplified using 5 U of Thermus aquaticus plemented with 1% BSA without serum in polypropylene (Taq) polymerase and primers specific for the 5′ end of IGF-IR: tubes (Fisher Scientific, Pittsburgh, PA, USA). ODN nts 1063–1082 (ACC ATT GAT TCT GTT ACT TC) or for the 5′ (100 mg/ml) were added at time zero, and 50% of the initial end of INS-R:19 nts 170–192 (GGA ACA ACC TCA CTA GGT dose was added 18 h later (final concentration ෂ26 ␮M). TGC A). ␤-actin mRNA was amplified simultaneously using Twenty-four hours after the first addition of ODN, cells were specific primers as described. Amplified products (10 ␮l) were plated in serum-free methylcellulose cultures without further electrophoresed on a 2% agarose gel, and transferred to a washing. nylon filter. Filters were pre-hybridized and probed with a 32P end-labeled ODN specific for the cDNA of IGF-I R (nts 1131– 1149) (CTG CTC CTC TCC TAG GAT GA), and INS-R Detection of phosphorylation of the IRS protein (CCT TGA GGT GAA CCA TCT CGA AGA TGA) (nts 409– 436). Hybridization was detected by autoradiography as The assay was performed essentially as described before.23,24 described.10–12,22 In brief, cells (107) were exposed to 5 mM of INS (Sigma), 100 ng/ml of IGF-I (R&D), or medium for 5 min at 37°C, lysed for 20 min in 1 ml ice-cold lysis buffer and centrifuged at Statistical analysis 15 000 r.p.m. The supernatants were preclared overnight at 4°C with protein A-sepharose (Sigma). The anti-IRS-2 antibody Arithmetic means and standard deviations were calculated on (kind gift of Dr Xiao Sun, Joslin Diabetes Center, Boston, MA, a MacIntosh computer using Instat 1.14 (GraphPad, San USA) was added, following by the protein A-sepharose to pre- Diego, CA, USA) software. Experiments were performed on Insulin and human hematopoiesis MZ Ratajczak et al 374 cells obtained from 11 healthy donors. Cells at each time low forward and orthogonal scatter analysis of the cells point were cultured in quadruplicate. Data were analyzed (Figure 2a). Within this region, cells were further analyzed for using the Student’s t-test for unpaired samples. Statistical co-expression of c-kit R and CD34 antigen (Figure 2b). Positiv- significance was defined as P Ͻ 0.01. ity was determined by comparison with isotype-matched antibody-labeled control cells. R1 delineates cells co-expressing both markers in the dual-labeled population (Figure 2c). Only Results those cells that satisfied R3 and R1 criteria were sorted and used in our experiments. The sorted population was Ͼ95% INS and IGF-I inhibit the apoptotic death of CD34+ pure (Figure 2d). cells To evaluate the influence of INS on survival of human early progenitors cultured under serum-free conditions, the CD34+, To examine the effect of INS and IGF-I on the growth and Kit-R+ cells sorted by FACS were subsequently cultured for survival of the early progenitor hematopoietic cells, we cul- 72 h in serum-free medium in the presence or absence of INS. tured the CD34+ cells for 7 days under serum-free conditions As shown in Table 2, the number of apoptotic cells was much in methylcellulose cultures in the presence of insulin greater in the cells cultured for 72 h in the absence of INS (20 ␮g/ml), IGF-I (50 ng/ml) or medium alone. We noted, that (P Ͻ 0.01). These data support our assumption, that the during this period of time, CD34+ cells did not proliferate even addition of INS to CD34+ Kit-R+ cells appeared to augment if exposed to the INS or IGF-I. the viability of hematopoietic progenitors by preventing them After 8 days in serum-free culture, cells were stimulated from undergoing apoptosis (Figure 3). This observation has with the following combinations of cytokines: KL+IL- also an important practical implication suggesting that INS 3+EpO+GM-CSF, EpO+KL, and IL-3+GM-CSF to obtain, should be added as an anti-apoptotic growth factor to the cul- respectively, CFU-Mix, BFU-E, and CFU-GM colonies. As ture media employed for growing and expanding human depicted in Table 1, the addition of INS significantly increased CD34+ cells. the survival of all progenitors tested, which were cultured for 7 days under serum free conditions (Table 1). Roughly, twice as many colony-forming cells were recovered when insulin was added to the serum-free culture medium. Similar results were obtained using IGF-1 (data not shown). Therefore, while Erythroid colony formation in serum-free, INS- INS and IGF-I did not stimulate the proliferation of human supplemented cultures CD34+ cells, both of these growth factors did enhance the survival of CD34+ progenitors cultured under serum-free conditions. Since both INS and IGF-I similarly enhanced the In contrast to serum supplemented cultures,4 EpO when used survival of the various progenitors evaluated in this study alone is unable to stimulate formation of human BFU-E under (CFU-Mix, BFU-E, CFU-GM), this pro-survival effect does not serum-free conditions.24 This difference most probably reflects seem to be lineage specific. the presence of biologically active concentrations of some Next we wondered if the pro-survival effect of INS is dose- growth factors present usually in the fetal serum component dependent. To address this issue, we cultured CD34+ cells in of the culture medium. Several reports suggested that INS serum-free medium in the presence of increasing doses of INS and/or IGF-I are required to costimulate with EpO erythroid (0–100 ␮g/ml). As demonstrated in Figure 1, the anti-apoptotic colony formation in serum-free cloning medium.5,8,17–19, 26–31 effect of INS becomes apparent at 1 ␮g/ml, and reaches a Nevertheless, we were not able to reproduce this observation maximum at 10–20 ␮g/ml. This concentration of INS signifi- when CD34+ cells were cultured in our serum-free culture sys- cantly improved the survival (P Ͻ 0.0001) of the human hem- tem. Therefore, we sought to identify cytokines which would atopoietic progenitors from all of the evaluated lineages. be able to co-stimulate with EpO the formation of BFU-E col- Further increases in INS concentration did not improve sur- onies under serum-free culture conditions (Table 3). There- vival of the CD34+ cells. fore, to determine the effect of different growth factors on To directly determine if INS effected the viability of progeni- BFU-E colony formation, CD34+ cells were plated in serum- tor cells, and to determine whether this enhancement occurs free methylcellulose containing EpO and INS, and various by inhibiting apoptotic death of these cells, highly enriched growth factors previously reported to augment erythropoiesis: in stem/early progenitor cells CD34+, Kit-R+ marrow cells were IGF-I, IGF-II, FGF-1, FGF-2, HGF, LIF, IL1␣, IL-1␤, IL-4, IL-6, isolated by FACS. Figure 2 shows results of a typical multi- IL-9, IL-11, G-CSF, GM-CSF, STK-1L, IL-3 and KL. parameter sort. The lymphocytic region, R3, was identified by We observed that the addition of most of these growth factors to EpO + INS did not stimulate BFU-E colony formation in our serum-free model (Table 3). The only exceptions were Table 1 Effect of insulin (INS) (20 ␮g/ml) on 7-day survival of KL, IL-3, GM-CSF and IL-9. KL was found to be the most + a CD34 progenitors in serum-free cultures potent stimulator of BFU-E growth under these culture conditions. IL-3, GM-CSF and IL-9 were only partially able to sub- − + Colonies No insulin ( INS) INS P value stitute for KL. The number and size of BFU-E colonies after stimulation with these growth factors were, however, always ± ± Ͻ CFU-Mix 10 4215 0.0001 smaller than after stimulation with KL, even when all these BFU-E 87 ± 25 154 ± 35 Ͻ0.0001 CFU-GM 119 ± 31 234 ± 49 Ͻ0.0001 growth factors were added to the cultures in combination (Epo + IL-3 + GM-CSF + IL-9) (data not shown). aResults are expressed as mean ± s.d. of colony counts from quad- The effect of KL and the other three factors was strictly EpO ruplicate cultures from four separate donors. Control cells were dependent because none of them was able to stimulate forma- held in serum-free medium for 7 days in the absence of insulin tion of BFU-E either alone or in combination (KL + IL-3 + GM- (no INS). CSF + IL-9; data not shown). Insulin and human hematopoiesis MZ Ratajczak et al 375

Figure 1 Effect of increasing doses of INS (0–100 ␮g/ml) on survival of clonogenic progenitors. Bone marrow CD34+ cells were cultured ﬁrst for 7 days under serum-free conditions, and at day 7 cells were stimulated with appropriate growth factors to grow CFU-Mix (a), BFU-E (b) and CFU-GM (c) colonies as described in the Materials and methods section. Data are pooled from three independent experiments performed on normal marrow cells, plated in quadruplicate.

Table 2 The effect of insulin (INS) on inhibiting apoptosis in the bone marrow CD34+ C-KIT-R+ A-T-BMMNCa

% alive cells % apoptotic cells

Without INS 63 ± 637± 6 INS added 20 ␮g/ml 82 ± 3b 18 ± 3b

aBone marrow CD34+ C-KIT-R+ A-T-BMMNC were cultured for 72 h without or in the presence of INS (20 ␮g/ml). The percentage of apoptotic cells was scored after staining with ‘in situ anti-apoptotic detection kit’ as described in Materials and methods. The experi- ment was repeated three times. bP Ͻ 0.01 as compared to cells unprotected with INS.

BFU-E colony formation. To clarify the role of INS in the formation of erythroid colonies, we next evaluated the influence of INS on erythroid colony formation by CD34+ cells cultured serum-free after stimulation with the mixture of EpO + KL or Epo + IL-3. We observed that the effect of INS on human erythroid colony formation was strongly dependent on the combination of growth factors used to costimulate BFU-E growth. It was big- Figure 2 Representative FACS analysis of bone marrow MNC dou- ger if the BFU-E cultures were stimulated sub-optimally ble-labeled for CD34 antigen (FITC) and c-kit receptor (PE). (a) For- (EpO + IL-3), and smaller if they were stimulated more opti- ward and side scatter analysis of adherent and T lymphocyte-depleted mally (EpO + KL). Accordingly, the number of colonies formed MNC. Lymphocyte region is defined by R3. (b) Analysis of cells dual- after stimulation with EpO + IL-3 and supplemented with INS labeled with FITC-anti-CD34 MoAb (anti-HPCA-2) and PE-anti-c-kit ± R (SR-1) MoAb. (c) The brightest dual-labeled cells are indicated by was approximately 30% higher as compared (145 45) to cul- R1. (d) Flow analysis of purity of sorted cell population. tures without INS (97 ± 24), and colonies were also larger in size (data not shown). By contrast, addition of insulin to EpO + KL had no significant effect on colony number Effect of exogenous INS on hematopoietic colony (304 ± 83 vs 299 ± 79) although colony size was increased in formation in serum-free cultures these cultures when INS was present. Since we have employed INS-free albumin for preparing As noted above, we found that INS if added alone to human our serum-free medium, the fact that even in the complete CD34+ cells cultured under serum-free conditions, did not absence of INS or IGF-I normally hemoglobinized erythroid stimulate their proliferation. Moreover, we found employing colonies developed after stimulation with EpO + KL or our serum-free culture system that the addition of both INS EpO + IL-3, indicates that neither INS nor IGF-I are required and/or IGF-I were insufficient to costimulate EpO-dependent for development of human erythroid colonies. Insulin and human hematopoiesis MZ Ratajczak et al 376

a b

c d

Figure 3 Effect of INS on apoptotic fate of CD34+, Kit-R+ MNC held in serum-free suspension cultures for 72 h. Fragmented DNA, as occurs in cells undergoing apoptosis, is detected by the presence of green nuclear fluorescence (ApopTag). (a and b) Corresponding phase and fluorescent photomicrographs of cells cultured with INS (20 ␮g/ml). Nuclei were counterstained with propidium iodide. No apoptotic cells are detected in this sample using Apoptag. (c and d). Corresponding phase and fluorescent photomicrographs of cells cultured without INS (20 ␮g/ml). Note the presence of apoptotic cells which stain positive for green fluoresence.

Table 3 Growth of BFU-E colonies from human bone marrow CD34+ cells plated in serum-free methylcellulose cultures containing Epo (5 U/ml) and INS (10 ␮g/ml), and other factors

BFU-E growth stimulated with: No. BFU-E colonies (± s.d.)/104 CD34+ cells plateda

EpO + INS no colonies EpO + INS + IGF-I no colonies EpO + INS + IGF-II no colonies EpO + INS + IL-1 ␣ no colonies EpO + INS + IL-1 ␤ no colonies EpO + INS + IL-4 no coloniesb EpO + INS + IL-6 no colonies EpO + INS + IL-11 no colonies EpO + INS + FGF-1 no colonies EpO + INS + FGF-2 no colonies EpO + INS + HGF no colonies EpO + INS + G-CSF no colonies EpO + INS + LIF no coloniesb EpO + INS + STK-1L no colonies EpO + INS + VEGF no colonies EpO + INS + PRL no colonies EpO + INS + IGF-I + IL-4 + IL-6 + IL-11 + FGF-2 + LIF no coloniesb EpO + INS + IL-9 11 ± 7 EpO + INS + GM-CSF 48 ± 36 EpO + INS + IL-3 118 ± 63 EpO + INS + KL 260 ± 63

aData are given as mean ± s.d. colony numbers in 24 cultures in six independent experiments. BFU-E were scored as colonies of Ͼ50 cells. bIncrease in small erythroid clusters of Ͻ50 cells.

Effect of delayed addition of insulin on growth of costimulate proliferation of the more differentiated erythroid CD34+ cells in serum-free cultures progenitors. Therefore, in order to determine whether insulin affects the terminal maturation of erythroid colonies, we The finding that INS and IGF-I can augment the size of BFU- examined BFU-E colony growth stimulated with EpO + IL-3, E colonies indicates that both these growth factors may when insulin (10 ␮g/ml) was either not added or added at day Insulin and human hematopoiesis MZ Ratajczak et al 377 0, 6 or 9 to the cultures (Table 4). Addition of insulin at day INS-R is expressed at all stages of hematopoiesis, whereas 6 fully restored the number of BFU-E colonies, and such col- IGF-IR is not detectable at the earliest progenitors but appears onies also achieved normal size compared to those grown in on the more differentiated cells. supplemented with insulin at day 0 serum-free cultures. Addition of IGF-I (50 ng/ml) at day 6 also restored the number of BFU-E colonies (data not shown). INS- and IGF-I-induced signal transduction in Similarly, when BFU-E colonies were grown in serum-free erythroid precursors medium stimulated with EpO + KL, addition of insulin at day 6 and 9 restored colonies to their normal size (data not A novel protein, insulin receptor substrate (IRS-2, 4PS) which shown). These data show that INS or IGF-I stimulate the is involved in the signaling by INS, IL-4 and probably other, proliferation and maturation of more differentiated erythroid similar factors, has been recently characterized.32 To provide progenitor cells. additional evidence that INS and IGF-I are able to stimulate differentiated erythroid cells, we analyzed BFU-E-derived cells for phosphorylation of the IRS-2 upon exposure to these fac- Expression of INS and IGF-I receptors on CD34+ early tors. As shown in Figure 8, INS as well as IGF-I, markedly human hematopoietic cells and more mature augmented phosphorylation of IRS-2 in the 9 day BFU-E hematopoietic cells isolated from BFU-E, and (obtained by culture in the presence of EpO + KL). The magni- CFU-GM colonies tude of this augmentation for INS was comparable to the one seen in the control, CHO cells transfected with human INS-R To learn more about the role of INS-R/INS, and IGF-I-R/IGF-I (CHO-INS-R), and somewhat less for IGF-I (3.8- and 2.2-fold receptor/ligand interactions in regulating human erythropo- increase above the value obtained with medium alone). More- iesis, we evaluated the expression of both INS-R and IGF-I-R over, an additional smaller (90 kDa) band consistent with the in human hematopoietic cells at different stages of develop- phosphorylated beta chain of INS-R and/or IGF-IR was noted ment. Expression of these receptors was evaluated at first in in the INS- IGF-I-stimulated cultures. These findings support the human bone marrow CD34+, c-kit-R+ cells sorted by the conclusion that maturing erythroid cells not only express FACS, according to the criteria depicted in Figure 2. RT-PCR both receptors, but are also responsive to INS and IGF-I analysis of INS-R and IGF-1R expression in the R3/R1 sorted stimulation. CD34+, c-kit R+ cells revealed mRNA for INS-R but not for IGF-IR mRNA (Figure 4a, c). Double staining with anti-CD34 and either anti-INS-R or IGF-I R antibody showed that all Effect of antisense ODN targeting IGF-IR on INS CD34+ cells expressed INS-R (Figure 4b), but not IGF-1R influence on formation of BFU-E colonies (Figure 4d) protein. Specificity of the antibodies used in our studies was confirmed as described in Materials and methods. Because IGF-IR was easily detectable in the more mature Next, expression of INS-R and IGF-1R was evaluated in the erythroid cells (Figures 6 and 7), it is uncertain whether INS more mature hematopoietic cells derived from CFU-GM and exerted its effect through its own receptor, INS-R, or structur- BFU-E colonies. For this purpose, CD34+ cells were plated in ally related IGF-I R.33 To address this question, we perturbed the serum free-medium and stimulated with the appropriate IGF-IR expression in BFU-E colonies with sequence-specific growth factors to form BFU-E or CFU-GM colonies. Individual antisense ODN. Downregulation of IGF-IR mRNA expression colonies were isolated at days 6 and 9, and analyzed by RT- (Figure 9), did not influence the number (mean ± s.d.) of BFU- PCR. mRNA for INS-R was detected in all samples (Figure 5). E stimulated with EpO + KL (240 ± 65 vs 242 ± 80) or EpO + In contrast to CD34+, c-kit R+ cells, IGF-1R mRNA was readily IL-3 (112 ± 39 vs 113 ± 36) that arose in the absence of pres- detectable in the cells isolated from day 6 BFU-E and CFU- ence of the IGF-IR antisense ODN, respectively. It is also GM colonies (Figure 6). important to note that none of the control ODN employed Similar results were obtained when cells isolated from day (sense, scrambled) affected colony formation (data not 6 and day 9 CFU-GM and BFU-E colonies were stained with shown). fluorochrome-conjugated anti-INS-R and anti-IGF-1R anti- These data show that INS is able to influence the maturation bodies. As shown in Figure 7, both INS-R and IGF-1R were of the erythroid progenitors stimulating its own, but not neces- easily detectable by FACS analysis on the cells isolated from sarily structurally related IGF-I R. day 6 BFU-E and CFU-GM colonies. These data suggest that

Table 4 Effect of insulin (10 ␮g/ml) on BFU-E formation by CD34+ Discussion MNC in serum-free methylcellulose culuresa We report here that the INS-R was detected on both mRNA + + Insulin BFU-E (±s.d.) and protein level on a highly enriched CD34 , c-kitR (104 cells/ml) stem/progenitor cell population, and on cells isolated from hemopoietic colonies. In contrast, IGF-IR was detected only None 103 ± 27 on the more mature cells isolated from day 6 BFU-E and CFU- Added day 0 144 ± 36b GM colonies. The fact that INS and IGF-I receptors are ± b Added day 6 145 41 expressed on erythroid cells was already suggested earlier by Added day 9 123 ± 43 other investigators who found that the cells isolated from BFU- E colonies bind radiolabeled INS and IGF-I.34 Our data aBFU-E were stimulated with EpO + IL-3. Data are given as mean ± s.d. colony numbers in 16 cultures in four independent experi- additionally indicate that IGF-IR expression in contrast to INS- ments. R expression may correlate positively with differentiation and bP Ͻ 0.01 as compared to the cultures grown without the presence maturation status of the hemopoietic cells. In agreement with of INS. this suggestion, it was recently reported that the IGF-IR Insulin and human hematopoiesis MZ Ratajczak et al 378

Figure 4 Expression of insulin (a) and IGF-1 (c) receptor mRNA by RT-PCR in sorted CD34+ c-kit R+ bone marrow MNC. lane 1, mRNA + + from CD34 c-kit R marrow MNC; lane 2, positive control (mRNA from A-T-BMMNC); lane 3,negative H2O control. (b) and (d) show results of double-labeling of bone marrow MNC with FITC-anti-CD34 and PE-anti-INS-R (b) or FITC-anti-CD34 and PE-anti-IGF-1R (d).

Figure 5 RT-PCR analysis of INS-R expression in bone marrow + + CD34 , KIT-R MNC (lanes 1 and 2), day 6 BFU-E (lane 3) and day Figure 6 RT-PCR analysis of IGF-IR expression in bone marrow 6 CFU-GM (lane 4), day 9 BFU-E (lane 5) and day 9 CFU-GM (lane CD34+, KIT-R+ MNC (lanes 1 and 2), day 6 BFU-E (lane 3) and day 6). Lane 7, negative H O control for the RT-PCR reaction. 2 6 CFU-GM (lane 4), day 9 BFU-E (lane 5) and day 9 BFU-GM (lane 6). Lanes 7 and 8 are negative controls for the RT-PCR reaction. expression increases on human B lymphocytes along with their maturation to the plasma cells.35–37 related INS-R by IGF-I.26,33 The observation that INS provides We also found that CD34+ cells if plated under serum-free only partial protection from apoptosis suggests that other fac- conditions, do not proliferate when stimulated with INS or tors play a role in survival of the hematopoietic progenitor IGF-I alone. However, addition of either of these growth fac- cells. These factors could include specific signals delivered by tors to serum-free medium appears to increase survival and other hematopoietic growth hormones and cytokines, or sig- viability of these cells. These findings, as well as our pre- nals generated after interaction of adhesion molecules viously published data10,11,22 demonstrate that receptors with expressed on early hematopoietic cells with their appropriate intrinsic tyrosine kinase activity might mediate a signal pre- ligands present in hematopoietic microenvironment. Most, or venting CD34+ cells from undergoing apoptosis. Moreover, specific combination(s) of these factors might be required for since IGF-IR was not detectable, in our hands, on CD34+ cells, the optimal survival of the CD34+ cells. We also noted with we can assume that the effect of IGF-I to inhibit apoptosis in interest that CD34+ cells stimulated with EpO + INS or CD34+ cells could be a result of stimulation of structurally EpO + IGF-I did not form BFU-E colonies in serum-free cul- Insulin and human hematopoiesis MZ Ratajczak et al 379

Figure 7 FACS analysis of INS-R (b, e) and IGF-IR expression (c, f) in cells from day 6 BFU-E (upper panels) and CFU-GM (lower panels) colonies isolated form serum-free cultures. (a) and (d) are cytograms of cells isolated from day 6 BFU-E (a) and CFU-GM (d) colonies. tures. This observation is in apparent discordance with pre- the structurally similar IGF-1R. Moreover, this weak erythro- viously published data,5,8,17–19,26–31 and could be partially poietic effect of INS, and IGF-I was observed even when INS explained by the fact that in our study we have been cloning or IGF-1 was added to the culture medium as late as day 6 bone marrow CD34+ cells, depleted from hematopoietic after plating. At this time point the growth of BFU-E colonies accessory cells. These latter cells could secrete KL or IL-3 is already established, and the cells present in BFU-E colonies and/or other factors which can co-stimulate growth of the do not express any more CD34 antigen. Moreover, we docu- BFU-E colonies. mented that INS and IGF-I activated signal transduction in From all the growth factors and cytokines we have tested, maturing human erythropoietic cells as determined by only KL, and to a much lesser degree IL-3, GM-CSF and IL- phosphorylation of the insulin receptor substrate-2 (IRS-2) 9, costimulated EpO-dependent growth of BFU-E. These data protein.32 All these data together suggest that both INS and confirm that KL is the major regulator of erythropoiesis. This IGF-I in contrast to KL exert their effects predominantly during extends our previous observation,10,11 that downregulation of the latter stages of erythropoiesis. A recent report that INS and c-kit R on human CD34+ cells completely inhibits BFU-E IGF-I stimulate the maturation and differentiation of cells from growth, even if EpO is present in the cultures.11 The fact that the erythroid lineage is in agreement with the data IL-3, GM-CSF and IL-9 are able to costimulate with EpO presented here.28 erythropoiesis to some degree may explain why profound A growing body of data suggests that the role of the insulin anemic w and sl mutant mice, which have defects in KL–c- growth factor family in the regulation of the early stages of kit R axis, still show some level of erythropoiesis.13 hemopoiesis might be over-stated. Accordingly, mice with We also evaluated the effect of INS and IGF-1 on the later deletional mutations of genes encoding IGF-I, IGF-2, IGF-R stages of erythroid colony formation. Both growth factors if and IRS-1,39–43 Laron dwarf patients with drastically reduced added to the cultures of CD34+ cells, which were stimulated IGF-I levels44 or acromegalic patients with elevated IGF-I lev- ‘suboptimal’ with EpO + IL-3, augmented slightly formation of els, all show normal erythropoiesis.45 Moreover, the individ- hemoglobinized colonies. This effect was not previously uals with an inherited absence of INS-R,46,47 and IGF-I48 were observed in our serum supplemented cloning system,12 which found to also be hematologically normal. Finally, the patients was probably ‘contaminated’ by INS present in the serum.8,38 treated with IGF-I for dwarfism or AIDS-related wasting syn- We also found, that when the IGF-1R expression was down- drome,49 similarly like diabetics receiving INS, do not show regulated using an antisense strategy, the effect of INS on the any signs of erythroid hyperplasia. late development of BFU-E colonies was still preserved. This We conclude therefore, that the role of the insulin growth suggests that INS acted through its own INS-R and not through factor family in regulating human erythropoiesis is probably Insulin and human hematopoiesis MZ Ratajczak et al 380 over-estimated in the literature. Our data also further strengthen the role of KL as the most critical regulator of BFU- E development. Without KL, BFU-E do not form colonies in serum-free medium even if supplemented with EpO and INS or IGF-I. In fact it was reported recently, that both EpO-R and c-Kit R signaling pathways are necessary for normal erythropoiesis.1–3 Of all the growth factors tested, only IL-3, GM-CSF and IL-9 could partially replace the KL function. We also demonstrated that, in contrast to KL, both INS and IGF-I are most important for late erythroid maturation, acting on cells more mature than CD34+. In ‘sub-optimal’ stimulatory conditions, both growth factors are able to increase proliferation of maturing erythroid precursors slightly, and speed up the process of their hemoglobinization. Nevertheless, even in the total absence of both insulin and IGF-I, EpO + KL are sufficient to stimulate complete development of BFU-E. Finally, our antisense data indicate that this weak erythropoietic effect of INS is mediated through its own receptor, and not through structurally related IGF-I R.33

Acknowledgements

This work was supported in part by grants from NIH (MZR) and the University of Pennsylvania Research Foundation (MAW). We are indebted to Dr Alan M Gewirtz for critical review of this manuscript and insightful comments.

References

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