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HEMATOPOIESIS
From the Department of Pathology and Laboratory
Medicine and the Department of Medicine, University of Pennsylvania
School of Medicine, Philadelphia, PA; and the Department of Medicine,
University of Alberta, and the Canadian Blood Services, Edmonton,
Alberta, Canada.
The aim of this study was to explore further the hypothesis
that early stages of normal human hematopoiesis might be coregulated by
autocrine/paracrine regulatory loops and by cross-talk among early
hematopoietic cells. Highly purified normal human CD34+
cells and ex vivo expanded early colony-forming
unit-granulocyte-macrophage (CFU-GM)-derived, burst forming
unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte
(CFU-Meg)-derived cells were phenotyped for messenger RNA
expression and protein secretion of various growth factors, cytokines,
and chemokines to determine the biological significance of this
secretion. Transcripts were found for numerous growth factors
(kit ligand [KL], FLT3 ligand, fibroblast growth factor-2 [FGF-2],
vascular endothelial growth factor [VEGF], hepatocyte growth factor
[HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin
[TPO]); cytokines (tumor necrosis factor- The development of hematopoietic cells is
regulated by hematopoietic growth factors, cytokines, and chemokines
secreted in the bone marrow (BM) microenvironment by accessory cells
(eg, fibroblasts, macrophages, osteoblasts, and endothelial cells) and
T lymphocytes (eg, as part of TH1- and
TH2-mediated responses),1-5 as well as by a
variety of other mechanisms.1-3 Recently, we and others
demonstrated the presence of messenger RNA (mRNA) for several
regulatory proteins in normal human BM CD34+
cells,6-12 megakaryocytes,11-14 and
mononuclear cells (MNCs)15 and suggested that an
intercellular cross-talk network and/or autocrine/paracrine regulatory
loops may play a role in the regulation of normal hematopoiesis.
In fact, positive regulatory loops mediated by interleukin 1(IL-1),
kit-ligand (KL), FLT3 ligand, and erythropoietin (EPO), and negative
ones mediated by transforming growth factor- On the basis of these findings, we hypothesized that normal
hematopoietic cells have developed mechanisms to communicate with each
other. To investigate this possibility, we phenotyped highly purified
normal human CD34+ cells and ex vivo expanded human
colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst
forming unit-erythroid (BFU-E)-derived, CFU-megakaryocyte
(CFU-Meg)-derived, and CFU-fibroblasts (CFU-F)-derived cells,
not only for mRNA expression of growth factors, cytokines, and
chemokines but also, and more importantly, by means of highly sensitive
enzyme-linked immunosorbent assay (ELISA)-based immunoassays, for the
presence of secreted proteins in media conditioned by these cells.
Furthermore, to better define the role these regulatory proteins play
in the biology of hematopoietic cells, we performed various functional
assays. Conditioned media harvested from normal CD34+ cells
were examined to see whether they would increase survival and/or
stimulate proliferation of other freshly isolated CD34+
cells, chemo-attract hematopoietic cells, and finally, stimulate endothelial cells.
We demonstrate, for the first time, that numerous growth factors,
cytokines, and chemokines are secreted at physiological concentrations
by normal human CD34+ cells and myeloid, erythroid, and
megakaryocytic precursor cells, and we provide evidence for the
existence of intercellular cross-talk networks and autocrine and/or
paracrine regulation of normal hematopoiesis.
Normal human hematopoietic cells and CD34+ selection by
fluorescence-activated cell sorting
Human myeloid, erythroid, and megakaryocytic precursor cells
Reverse transcriptase-polymerase chain reaction
Enzyme-linked immunosorbent assay The secretion of growth factors, cytokines, and chemokines by hematopoietic cells was evaluated by Quantikine human immunoassays (all from R&D) according to the manufacturer's protocol. Equal numbers (1 × 106/mL) of CD34+ cells or normal human CFU-GM-, BFU-E-, CFU-Meg-, and BM stroma-derived cells that had been cultured for 24 hours in serum-free conditions prior to the harvesting of the conditioned media were used. The collected media were analyzed by a quantitative sandwich enzyme immunoassay technique. The sensitivity of the ELISA assays was as follows: hepatocyte growth factor (HGF), greater than 40 pg/mL; vascular endothelial growth factor (VEGF), greater than 5 pg/mL; TPO, greater than 15 pg/mL; fibroblast growth factor-2 (FGF-2), greater than 3 pg/mL; TGF- 1, greater than
31 pg/mL; TGF-2, greater than 31 pg/mL; GM-CSF, greater than 7 pg/mL; insulin-like growth factor-1 (IGF-1), greater than 12 pg/mL; FLT3 ligand, greater than 7 pg/mL; KL, greater than 3 pg/mL; IL-16, greater than 31 pg/mL; macrophage inflammatory
protein-1 (MIP-1), greater than 31 pg/mL; MIP-1, greater than
31 pg/mL; monocyte chemoattractant protein-1 (MCP-1), greater
than 31 pg/mL; platelet factor-4 (PF-4), greater than 10 pg/mL; and IL-8, greater than 1 pg/mL.
Chemotaxis studies All experiments were performed in triplicate on BM MNCs, highly purified CD34+ cells, or serum-free expanded myeloid, erythroid, or megakaryocytic cells. Briefly, after isolation, the cells were resuspended in serum-free medium (106/mL) and equilibrated for 10 minutes at 37°C. In the meantime, 600 µL per point of prewarmed serum-free conditioned medium was added to the lower chamber of the Costar Transwell 24-well plate, 6.5 mm diameter, 5 µM pore filter (Costar Corning, Cambridge, MA). Subsequently, 100 µL aliquots of the cell suspension were distributed to the upper chambers, and cultures were incubated at 37°C, 95% humidity, 5% CO2, for 4 hours. The plates were evaluated under an inverted microscope: cells from the lower chambers were collected, and cell number was scored by means of FACScan (Becton Dickinson) as described.22 The cells were gated according to their forward-scatter characteristics (FSCs) and side-scatter characteristics (SSCs) and counted during a 20-second acquisition. The results of chemotaxis of the BM MNCs were recorded by cytometer and shown as the number of events registered in the particular gates. In some chemotaxis experiments, in which highly purified CD34+ cells and BFU-E-, CFU-GM-, and CFU-Meg-derived cells were employed, the results are presented as a migration index: the ratio of (1) the number of cells that migrated toward the conditioned medium that was being tested to (2) the number of cells that migrated toward the serum-free culture medium.Effects of CD34+ cell-conditioned media on survival and proliferation of CD34+ cells Purified CD34+ cells were seeded at a density of 1 to 5 × 105/mL in serum-free medium not conditioned (see above) or conditioned by CD34+ cells (2 × 106/mL per 24 hours). After 7 days, the number of living cells was evaluated by the 0.5% trypan blue exclusion test, and the cells were plated in methylcellulose or plasma clot cultures and stimulated to grow CFU-GM, BFU-E, and CFU-Meg colonies. Briefly, CD34+ cells (1 × 104) were cloned in 1 mL Iscoves modified Dulbecco medium supplemented with artificial serum as described above.19-22 Recombinant human growth factors appropriate to the colonies to be grown were added to the mixture, which was then transferred to 3.5-cm plastic petri dishes and incubated (37°C, 95% air, 5% CO2, in a humidified atmosphere). The growth factors and concentrations used were EPO (5000 IU/L) plus KL (100 ng/mL) for BFU-E; IL-3 (20 U/mL) plus GM-CSF (5 ng/mL) for CFU-GM; and TPO (50 ng/mL) for CFU-Meg. Colonies were counted under an inverted microscope on day 11 for CFU-GM and CFU-Meg and on day 14 for BFU-E colonies.Activation of caspase-3 was determined by FACS through the employing of intracellular staining with a monoclonal antibody that recognizes the activated form of caspase-3 according to the manufacturer's protocols (BD Pharmingen, San Diego, CA). Effects of rh factors on survival and proliferation of CD34+ cells We isolated 1 × 105 CD34+kit-R+ MNCs by FACS and then resuspended them in Iscove's DMEM containing the serum-free supplement described previously. The cells were cultured in 6-well plastic plates in the presence (or absence) of rh insulin (20 µg/mL), rhVEGF (100 ng/mL), rhHGF (100 ng/mL), rhFGF-2 (100 ng/mL), rhIGF-1 (100 ng/mL), rhKL (100 ng/mL), and rhFLT3 ligand (100 ng/mL) for 72 hours (37°C, 95% humidity, 5% CO2) and then washed 1 × in PBS before fixation in 4% neutral-buffered formalin. After fixation, the cells were sedimented by gravity onto slides covered with Cell-Tak tissue adhesive (Collaborative Biomedical Products, Bedford, MA). Apoptosis was detected in these cells by means of the in situ apoptosis detection kit Apoptag (Oncor, Gaithersburg, MD) according to the manufacturer's protocol as described.20,25 In this assay, terminal deoxynucleotide transferase was used to label the fragmented ends of DNA that have undergone apoptosis with digoxigenin-deoxyuridine triphosphate. The digoxigenin label was subsequently labeled with anti-digoxygenin antibody, which allowed simple, direct enumeration of apoptotic cells with a fluorescence microscope. Activation of caspase-3 was determined by FACS according to the manufacturer's protocol (BD Pharmingen).Proliferation of human umbilical vein endothelial cells Human umbilical vein endothelial cells (HUVECs) were isolated as previously described in detail.26 The cells were seeded 24 hours before the experiment in 96-well plates coated with fibronectin in growth medium (M199, 90%; fetal calf serum, 10%; bovine brain extract, 1 mg/mL; human epidermal growth factor, 1 ng/mL; hydrocortisone, 1 mg/mL; heparin, 10 U/mL) at a density of 2.5 × 103. The next day, the wells were washed 3 × with sterile, prewarmed PBS, and subsequently we added 100 µL prewarmed growth medium, serum-free medium, or serum-free medium conditioned by CD34+ cells (the last was obtained by culturing CD34+ cells at 2 × 106/mL for 24 hours in serum-free medium). On days 2 and 4, the media were replaced by fresh ones. On day 6, the proliferative status of the cells was evaluated by means of a colorimetric assay (Celltiter 96; Promega, Madison, WI) according to the manufacturer's protocol. Briefly, 20 µL Aqueous One Solution was added to each well, and the plate was incubated for 4 hours at 37°C in a humidified, 5% CO2 atmosphere. The plate was then read at 490 nm by means of a 96-well plate reader, and the background absorbance of the culture medium was subtracted to yield the correct absorbance.Statistical analysis Arithmetic means and SDs were calculated on a MacIntosh computer with Instat 1.14 (Graphpad, San Diego, CA) software. Data were analyzed by means of the Student t test for unpaired samples. Statistical significance was defined as P < .05.
Numerous growth factors, cytokines, and chemokines are expressed by normal human BM and mPB CD34+ cells, erythroblasts, megakaryoblasts, and myeloblasts The expression of mRNA for various hematopoietic growth factors, cytokines, and chemokines in normal human highly purified BM and mPB CD34+ cells and CFU-GM- (CD33+), BFU-E- (GPA-A+), CFU-Meg- (CD41+), and CFU-F-derived cells is shown in Table 2. The purity of these cells is shown in Figures 1 and 2.
The hematopoietic growth factors we selected for these studies are
known to be important regulators of mesodermal development and to
activate receptors possessing intrinsic tyrosine kinase activity. We
found that both BM- and mPB-derived CD34+ cells express
mRNA for KL, FLT3 ligand, FGF-2, VEGF, HGF, and IGF-1 (Table 2), but
not for macrophage CSF or NGF-
We also demonstrated mRNAs for chemokines such as MIP-1 Numerous growth factors, cytokines, and chemokines are secreted by CD34+ cells, erythroblasts, megakaryoblasts, and myeloblasts Hence, our next step was to attempt to correlate these RT-PCR findings with protein secretion, using sensitive, commercially available ELISA assays. We found that normal human BM-derived CD34+ cells, myeloblasts, erythroblasts, megakaryoblasts, and BM fibroblasts secrete detectable amounts of various hematopoietic growth factors, cytokines, and chemokines (Tables 3, 4, and 5). These various regulatory proteins were secreted by highly purified CD34+ cells at picogram levels, which could potentially affect their proliferation either by stimulating (eg, KL, FLT3 ligand, and TPO) or inhibiting it (eg, TGF-1, TGF-2, and PF-4). Further, these secreted
proteins (KL, FLT3 ligand, TPO, and IGF-1) could autoprotect
CD34+ cells from undergoing apoptosis. Moreover, proteins
such as VEGF, HGF, FGF-2, and IL-8 could attract and/or stimulate human
endothelial cells. We confirmed our previous observations that
CD34+ cells secrete several HIV-related -chemokines that
could protect these cells from infection by R5 (macrophagotropic)
HIV,11,12 and we report here, for the first time, that
highly purified human CD34+ cells secrete IL-16, another
factor that may interfere with HIV infection.28
We also observed that secretion of VEGF by CFU-GM-, BFU-E-, and
CFU-Meg-derived cells was significantly higher (eg, up to 820 pg/mL by
BFU-E-derived cells) than by CD34+ cells (20 pg/mL). In
addition, while cells from erythroid and megakaryocytic lineages
secreted high levels of TGF- Further, we found that human CD34+ cells secrete various
chemokines, such as MIP-1 Furthermore, CFU-Meg-derived cells secreted RANTES, PF-4, and IL-8 at
higher levels than CD34+ or stromal cells. MCP-1 was
secreted only by BM fibroblasts, and we did not detect the presence of
MCP-1, RANTES, MIP-1 Since normal human endothelial cells secrete several cytokines and
growth factors,31 we evaluated whether the
CFU-GM-, BFU-E-, and CFU-Meg-derived cells used in this study were
contaminated by endothelial progenitors (AC133+
and VEGF-R2+) and endothelial cells (AC133 Media conditioned by CD34+ cells attract other CD34+ cells, myeloblasts, and megakaryocytes To assess the biological significance of these regulators secreted by normal human BM CD34+ cells, we used transwell chemotaxis assays to examine whether conditioned media harvested from these cells attract other hematopoietic cells. To do this, we first evaluated the chemotactic potential of human BM MNCs toward media conditioned by CD34+ cells. Cells showing chemotaxis were collected, counted, and evaluated by FACS according to their FSC and SCC criteria. To our surprise, we found that normal human CD34+ cells secrete chemotactic factors that strongly attract primary human BM MNCs from the monocytic and granulocytic gates (Figure 4C,D).
When we extended these chemotaxis experiments to other precursors
(CFU-GM-, CFU-Meg-, and BFU-E-derived cells), we found that conditioned media harvested from normal human CD34+ cells
attracted normal myeloblasts and megakaryoblasts but not erythroblasts
(Figure 5A). Contrary to our
expectations, conditioned media harvested from normal CD34+
cells were also able to attract other CD34+ cells freshly
isolated from human BM (Figure 5A). This observation is intriguing as
SDF-1, recognized as a strong chemo-attractant of human
CD34+ cells,31-34 is not expressed in these
cells (Table 2), suggesting that another potent, as yet undiscovered,
chemo-attractant exists, and is present in media conditioned by
CD34+ cells.
Finally, since many potential chemo-attractants are secreted by CFU-F-derived fibroblasts, we evaluated whether media conditioned by these cells attract normal human myeloblasts, megakaryoblasts, erythroblasts, and CD34+ cells. In contrast to media conditioned by CD34+ cells, we found that media conditioned by BM fibroblasts attracted CD34+ cells only (Figure 5B). Media conditioned by CD34+ cells stimulate proliferation and inhibit apoptosis of CD34+ cells Having detected in conditioned media harvested from normal CD34+ cells the presence of several factors that may potentially increase cell viability (KL, FLT3 ligand, TPO, and IGF-1) or decrease it (TGF-1 and TGF-2), we decided to determine which
biological effect (stimulatory or inhibitory) would prevail when
freshly isolated CD34+ cells were cultured in media
conditioned by other CD34+ cells. To address this question,
normal BM CD34+ cells were cultured for 7 days in medium
conditioned (or not) by CD34+ cells. At day 7, the cells
were counted and their viability was determined by 0.5% trypan blue
exclusion tests; clonogenicity was evaluated in methylcellulose
cultures. Activation of caspase-3 in these cells was studied by FACS by
means of intracellular staining with monoclonal antibodies that
recognize the activated form of caspase-3.
We found that conditioned media harvested from CD34+ cells
slightly stimulated the proliferation of freshly isolated
CD34+ cells cultured for 7 days (data not shown) and
significantly improved their survival (Figure
6A). We also found in methylcellulose replating experiments that more lineage-specific progenitor cells, such
as BFU-E, CFU-GM, and CFU-Meg, retained their proliferative potential
if they were cultured for 7 days in media conditioned by
CD34+ cells (Figure 6B-D). Interestingly, this effect
depended on the concentration of CD34+ cells employed as
"target cells" in our assay. We found that if we cultured our
target population of CD34+ cells at low concentration
(1 × 105/mL), the protective effect of conditioned media
harvested from CD34+ cells at 2 × 106/mL per
24 hours was more evident than if we cultured the target population of
CD34+ cells at a concentration of 5 × 105/mL
(data not shown). It is obvious that in the latter case
CD34+ cells were able to enrich their culture medium much
faster with endogenously secreted factors. To further demonstrate that
biologically active factors are secreted by CD34+ cells, we
cultured 2 sets of samples of CD34+ cells
(5 × 105/mL) for 7 days in serum-free medium and every
24 hours gently spun down the cells in both sets. While the cells in
the first set were always resuspended in fresh medium, the cells in the second set were resuspended in the "old" (ie, conditioned) medium. We found that the viability of cells cultured for 7 days in the old
medium decreased by only 23% ± 6% compared with the cells that
were resuspended every day in the fresh medium, which decreased by
58% ± 7%. Thus, these data show that CD34+ cells
secrete factors that may improve their survival, at least under the
culture conditions employed in this work.
To better demonstrate the effect of media conditioned by CD34+ cells in preventing the apoptosis of other CD34+ cells, we also examined activation of caspase-3 (using intracellular FACS staining) in CD34+ cells cultured in fresh medium vs medium conditioned by CD34+ cells. We found that after 72 hours, caspase-3 became activated in 36% ± 7% of CD34+ cells cultured in serum-free medium, compared with no activation in medium conditioned by CD34+ cells (Figure 6E). Survival and proliferation of human CD34+ cells is distinctly regulated by various endogenously secreted growth factors added in recombinant form Having determined that normal human CD34+ cells endogenously secrete KL, FLT3 ligand, IGF-1, TPO, VEGF, FGF-2, and HGF, we set out to determine the effect these regulators have on apoptosis and/or proliferation of human CD34+ cells. To address this issue, we cultured human CD34+kit+ cells (isolated from BM MNCs by FACS to a purity greater than 98%) under serum-free conditions with each of the growth factors added exogenously and, after 72 hours, counted the total number of cells and the number of apoptotic cells in these cultures. We found that exogenously added rhKL, FLT3 ligand, TPO, and IGF-1, but not VEGF, FGF-2 and HGF, inhibited apoptosis in CD34+kit+ cells (Table 5). Moreover, as we expected, the presence of KL, FLT3 ligand, or TPO in a culture slightly stimulated the proliferation of CD34+kit+ cells. In contrast, exogenously added VEGF, FGF-2, and HGF had no such effect (Table 5).Media conditioned by CD34+ cells and CFU-GM-, CFU-Meg-, and BFU-E-derived cells stimulate proliferation of HUVECs Using the ELISA assay, we detected the presence of the angiopoietic factors VEGF, FGF-2, HGF, and IL-8 in conditioned media harvested from human CD34+ cells. Similarly, VEGF was found in the conditioned media harvested from CFU-GM, CFU-Meg, and BFU-E cells (Table 3). To determine whether these media affect the biology of endothelial cells, we cultured HUVECs in serum-free media conditioned or not conditioned by human CD34+ cells and CFU-GM-, CFU-Meg-, or BFU-E-derived cells (Figure 7). We found that media conditioned by all these cells increased the survival of HUVECs and stimulated their proliferation (Figure 7).
The involvement of autocrine/paracrine regulatory axes has been
proposed in the growth of many solid tumors35 and
leukemias,36-39 as well as normal hematopoietic
stem/progenitor cells.6-10,40-42 We and others have shown
that normal human CD34+ cells express mRNA for several
hematopoietic regulators (KL, FLT3 ligand, EPO, IL-1, and
TGF- Advances in cell isolation and culture techniques have made it possible to obtain highly purified cells, such as human CD34+ cells, myeloblasts, megakaryoblasts, and erythroblasts, for research purposes.3,19,44 Moreover, sensitive ELISA immunoassays now permit detection of picogram levels of hematopoietic growth factors, cytokines, and chemokines. Hence, after finding that highly purified normal human CD34+ cells and early CFU-GM-, BFU-E-, CFU-Meg-, and CFU-F-derived cells express mRNA for various growth factors, cytokines, and chemokines, we went on to perform extensive studies to evaluate whether these factors are in fact secreted by these cells. In this study, we reconfirmed that normal CD34+ cells
express mRNA for KL, FLT3 ligand, IL-1, IL-8, and
TGF- To better understand the biological significance of our findings, we
examined whether conditioned media harvested from CD34+
cells promote proliferation and survival of freshly isolated CD34+ cells, chemo-attract other CD34+ cells or
more mature CFU-GM-, BFU-E-, and CFU-Meg-derived cells, and
stimulate the proliferation of endothelial cells (HUVECs). We found
that conditioned media harvested from CD34+ cells
stimulated proliferation slightly and inhibited apoptosis of
freshly isolated BM CD34+ cells, a fact that could be
explained by the presence of KL, FLT3 ligand, TPO, IGF-1, and IL-1
proteins in these media. Since CD34+ cells were also found
to secrete inhibitors, such as TGF- We are aware, however, that the ELISA assays were performed on a population of CD34+ cells that remains heterogeneous even after purification, containing, in addition to hematopoietic stem/progenitor cells, endothelial,31,49,50 stromal,51 and lymphoid52 progenitor cells. Nevertheless, our finding of many hematopoietic regulators detectable in conditioned media harvested from human CD34+ cells may have important implications not only for understanding the biology of hematopoietic stem cells but also for therapeutic purposes. It has recently been postulated that human stem cells proliferate and self-renew if exposed to high concentrations of growth factors and, in contrast, differentiate at low concentrations.5 Thus, the presence of hematopoietic stimulators endogenously secreted by CD34+ cells at low concentrations, as shown by us, could explain the difficulty experienced in achieving ex vivo expansion of human hematopoietic stem cells. The fact that media conditioned by CD34+ cells were found to attract other hematopoietic cells is intriguing. However, although CD34+ cells were shown to secrete many chemokines, SDF-1, which is known to be an important chemo-attractant of CD34+ cells,31-34 was not expressed in these cells. Hence, we suggest that chemo-attractants for human CD34+ cells other than SDF-1 exist and are secreted by normal human CD34+ cells. Currently, we are working on identifying these factors. We are also phenotyping lymphoid cells, which are attracted by human CD34+ cells, in an attempt to identify the subsets of lymphocytes that may co-regulate proliferation of hematopoietic cells.4 Finally, in our study, the stimulation of endothelial cells (HUVECs) by media conditioned by CD34+ cells correlated with the secretion of VEGF, FGF-2, HGF, and IL-8 proteins by CD34+ cells, which indicates the existence of cross-talk between normal hematopoietic progenitors and endothelium.53-55 Proliferation of HUVEC cells was also stimulated by conditioned media harvested from CFU-Meg-, BFU-E-, and CFU-GM-derived cells. Hence, we postulate here that, like leukemic cells,17,56-59 normal hematopoietic cells possess a mechanism allowing them to stimulate the proliferation of endothelium. We believe that this may be a common mechanism to secure vascularization and an appropriate supply of blood to the areas of intensive hematopoiesis.53,54 The secretion of various chemokines by normal human CD34+
cells observed in this study is also intriguing and has important consequences for cell biology. In agreement with this, we recently reported that the Many growth factors, cytokines, and chemokines were also expressed and secreted by more differentiated cells and might potentially play an important role in the cross-talk between different subsets of hematopoietic cells.61 However, defining the precise role these factors play in CFU-GM, CFU-Meg, or BFU-E development will require more study. Interestingly, we also found that medium conditioned by CFU-F-derived fibroblasts attracts normal human CD34+ cells but does not attract the more differentiated cells, such as myeloblasts, megakaryoblasts, and erythroblasts. These observations suggest that the chemotaxis of hematopoietic cells to stroma is regulated developmentally and explains why only early CD34+ cells home to BM hematopoietic niches.1,3 One of the putative candidates for this effect is SDF-1 secreted by BM stromal cells.31-34 The endogenously secreted factors we describe (and presumably those as
yet undiscovered) may play a key role in the biology of early
hematopoietic cells in vivo. In vitro, where recombinant molecules are
used, this effect will probably differ, especially when one considers
that endogenously secreted factors are very often much more
biologically effective than recombinant proteins.62 As an
example, autocrine TGF- Finally, it will be important to evaluate secretion of these factors by hematopoietic cells derived from the patients suffering from many and various hematological diseases, eg, cytopenias, myelodysplastic syndromes, and myeloproliferative disorders. When we develop a better understanding of intercellular cross-talk in normal and pathological states, we may target the various mechanisms regulating synthesis of these endogenous factors for new pharmacological approaches. In conclusion, we provide the first evidence that normal human BM or PB CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts secrete numerous growth factors, cytokines, and chemokines that contribute to intercellular cross-talk networks and regulate various stages of hematopoiesis. We believe that our observations open a new area of investigation into hematopoiesis.
The authors thank Dr D. Cines (Department of Pathology & Laboratory Medicine, University of Pennsylvania) for HUVECs, A. Dobrowsky and K. Esler for technical assistance, and Dr Mitch Weiss (CHOP, Philadelphia) for comments.
Submitted September 1, 2000; accepted January 25, 2001.
Supported by National Institutes of Health grant R01 HL61796-01 (M.Z.R.), the Leukemia and Lymphoma Society grant 64907-00 (M.Z.R.), and Canadian Blood Services R & D grant XE0004 (A.J.-W.).
M.M. and A.J.W. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented at the 41st Annual Meeting of the American Society of Hematology, New Orleans, LA, December 3-7, 1999, and published in abstract form in Blood. 1999;94(suppl 1):465a. Reprints: Mariusz Z. Ratajczak, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, University of Pennsylvania, 405A Stellar Chance Labs, 422 Curie Blvd, Philadelphia PA 19104; e-mail: mariusz{at}mail.med.upenn.edu.
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J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism Blood, February 15, 2002; 99(4): 1117 - 1129. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
T
20°C. The RNA pellet was washed in 75% ethanol and resuspended in
3 × autoclaved H2O. Subsequently, mRNA (0.5 µg) was
reverse-transcribed with 500 U Moloney murine leukemia virus reverse
transcriptase and 50 pmol of an oligodeoxynucleotide primer
complementary to the 3' end of the reported gene sequences of the
appropriate growth factors and cytokines. The resulting complementary
DNA (cDNA) fragments were amplified (for 30 cycles) by means of 5 U
Thermus aquaticus (Taq) polymerase and primers specific for the 5' end
of the gene sequences of the detected growth factors and cytokines.
Amplified products (10 µL) were electrophoresed on a 2% agarose gel
and transferred to a nylon filter and further documented
photographically. The sequences of primers employed in this study are
shown in Table 
comparison to other cytokines and growth factors.
Leukemia.
1998;12:371-381