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HEMATOPOIESIS
From the Department of Stem Cell Biology, Institute of
Laboratory Medicine, University Hospital of Lund, Lund, Sweden, and the
Department of Oncology, The Norwegian Radium Hospital, Oslo, Norway.
Hematopoietic stem cell (HSC) fate decisions between self-renewal
and commitment toward differentiation are tightly regulated in vivo.
Recent developments in HSC culture and improvements of human HSC assays
have facilitated studies of these processes in vitro. Through such
studies stimulatory cytokines critically involved in HSC maintenance in
vivo have been demonstrated to also promote HSC self-renewing divisions
in vitro. Evidence for negative regulators of HSC self-renewal is,
however, lacking. Tumor necrosis factor (TNF), if overexpressed, has
been implicated to mediate bone marrow suppression. However, whether
and how TNF might affect the function of HSC with a combined myeloid
and lymphoid reconstitution potential has not been investigated. In the
present studies in vitro conditions recently demonstrated to promote
HSC self-renewing divisions in vitro were used to study the effect of
TNF on human HSCs capable of reconstituting myelopoiesis and
lymphopoiesis in nonobese diabetic-severe combined immunodeficient
(NOD-SCID) mice. Although all cord blood and adult bone marrow
CD34+CD38 The output of millions of blood cells per second in
adult humans is strictly dependent on the presence of multipotent
short- and long-term reconstituting hematopoietic stem cells (HSCs). HSC fate decisions between self-renewal and commitment toward differentiation or potentially apoptosis are tightly regulated in vivo,
although the molecular mechanisms regulating these decisions remain
unknown.1 However, through gene targeting studies, several growth stimulatory cytokines have been demonstrated to be critically involved in HSC maintenance in mice.2-4 The potential role
of negative regulators of HSC maintenance in steady-state hematopoiesis or conditions characterized by hematopoietic failure is less clear.
Tumor necrosis factor- There are 2 main reasons why studies of human HSC self-renewal in vitro
have been difficult to pursue until recently. First, previously used in
vitro culture conditions appeared inefficient at promoting growth of
candidate human HSCs, and studies in mice suggested that such
conditions were usually associated with loss of HSC
potential.14-16 Second, the lack of functional assays for human HSCs addressing their ability to reconstitute myelopoiesis and
lymphopoiesis made it difficult to access HSC potential. The recent
cloning and characterization of the early acting cytokines c-kit ligand
(KL),2 flt3 ligand (FL),2 and thrombopoietin (Tpo),17 demonstrated to be critically involved in HSC
maintenance in mice,2-4,18,19 have dramatically improved
the ability to promote in vitro growth of candidate human
HSCs.20-23 More importantly, the use of these cytokines
combined with other developments in HSC culture conditions and the
ability to specifically track HSC divisions in vitro, have demonstrated
that HSCs undergoing cell divisions under such conditions can preserve
their HSC function.22-26 The evidence for this is
particular strong in mice, in which pluripotent long-term repopulating
HSCs can be directly evaluated.1 Through such studies,
multiple cytokines (including KL, FL, Tpo, and interleukin-11 [IL-11]) have been demonstrated to efficiently promote in vitro murine HSC self-renewing divisions.23,26,27 The recent
development and improvement of assays for candidate human HSCs have now
allowed corresponding studies in humans28-31 and
development of conditions, similar to those used in mice, which appear
to promote self-renewing divisions of candidate human
HSCs.23,25,32-34 Although it remains to be established how
predictable these assays are for long-term repopulating HSC activity,
the in vivo xenograft models appear particularly useful because they
selectively detect very primitive progenitor/stem cells capable of in
vivo reconstituting both myelopoiesis and
lymphopoiesis.30,31,35 Thus, the stage has been set for meaningful studies of regulation of self-renewal of candidate human HSCs.
In the present studies we used in vitro conditions recently
demonstrated to efficiently promote proliferation of murine and candidate human HSCs with sustained HSC function,23,26 to
address the potential effect of TNF on self-renewal of cells capable of repopulating myelopiesis and lymphopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Whereas all candidate HSCs
were capable of undergoing cell divisions in the presence of TNF, they
were dramatically compromised in their ability to reconstitute NOD-SCID
mice in vivo and long-term cultures in vitro. Rather, the presence of
TNF appeared to promote HSC differentiation. Thus, TNF appears to
negatively regulate maintenance of cycling human HSCs.
Isolation of CD34+ and
CD34+CD38 CD34+CD38 Hematopoietic growth factors, TNF molecules, and
antibodies
[3H]TdR incorporation assay for Jurkat cells
Ex vivo expansion cultures The CD34+ CB cells, CD34+CD38 CB cells, or
CD34+CD38 BM cells were cultured for 2 to 12 days in serum-free (SF) medium, either X-vivo 15 (Bio-Whittaker;
Walkersville, MD) supplemented with 1% detoxified bovine serum albumin
(BSA; Stemcell Technologies; Vancouver, BC, Canada) or Iscoves modified
Dulbecco medium (IMDM; Bio-Whittaker) supplemented with 20 mg/mL BSA,
10 µg/mL human insulin, and 200 µg/mL of human transferrin (BIT;
Stemcell Technologies) as well as 40 mg/mL low-density lipoprotein
(LDL; Sigma; St Louis, MO). CD34+ CB cells were seeded at
10 000 to 40 000 cells/mL, and CD34+CD38 CB
and BM cells at 800 to 3500 cells/mL. Cells were cultured (in the
absence or presence of TNF) in a cocktail of cytokines (rhKL 100 ng/mL,
rhFL 100 ng/mL, rhTpo 100 ng/mL, and rhIL-3 20 ng/mL; KFT3) based on
previous studies suggesting that these cytokines efficiently induce
proliferation of candidate HSCs with sustained hematopoietic
potential.23,26 Following culture, cells were enumerated
and evaluated functionally in long-term cultures or transplanted into
NOD-SCID mice. In some experiments cultured cells were also examined
with regard to apoptosis and cell-cycle and differentiation status.
Limiting dilution assay To investigate the direct effects of TNF on candidate stem cells, CD34+CD38 cells were seeded in
Terasaki plates (Nunc; Kamstrup, Denmark) at a density of 1 cell/well
in SF medium (X-vivo 15 and 1% BSA), supplemented with a cocktail of
cytokines (rhKL 50 ng/mL, rhFL 50 ng/mL, rhTpo 50 ng/mL, and rhIL-3 20 ng/mL) in the absence or presence of TNF 20 ng/mL. Wells (120/group)
were scored for cell growth following 10 to 12 days of incubation at
37°C in a humidified atmosphere with 5% CO2 in air.
Because the statistical chance (based on Poisson probability
distribution) of a well not receiving any cell is 37% by this method,
the maximum expected clones were 76.23
To compare recruitment of KFT3-cultured CB
CD34+CD38 NOD-SCID repopulating assay The NOD/LtSz-SCID mice (originally from The Jackson Laboratory, Bar Harbor, ME) were bred and housed under sterile conditions in microisolator cages and given irradiated food and acidified, autoclaved water. Mice were irradiated with 350 cGy from a 137Cs source at 8 to 12 weeks of age and given prophylactic ciprofloxacin 100 mg/L in the drinking water until analysis (6 weeks after transplantation). Tail vein transplantation/injection of hematopoietic cells suspended in 0.5 to 1.0 mL medium was performed within 12 hours of irradiation. In some experiments 1 × 106 irradiated (1500 cGy) accessory cells (MNC BM or CB cells or CD34+-depleted CB cells) were coinjected. After 6 weeks mice were killed by asphyxiation with CO2; femora and tibiae were collected, and engraftment was investigated by flow cytometric analysis (FACSCalibur) as described previously.35,37,38 Briefly, BM cells were counted and blocked with anti-mouse Fc-block (Pharmingen, San Diego, CA) and whole-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Subsequently, cells were stained with FITC-conjugated anti-human CD45 and CD71 Abs (Becton Dickinson) as well as anti-mouse CD45.1 (Ly 5.1)-PE Ab (Pharmingen). BM cells from untransplanted mice (negative controls) and mixtures of 0.1% to 0.5% human cells in murine BM (positive controls) were always included. If engraftment was detected by CD45/CD71 analysis (detection level 0.05%), lineage analysis with anti-HuCD34-FITC (progenitors), anti-HuCD19-PE (B cells; Becton Dickinson), or anti-HuCD15-PE and anti-HuCD66b-FITC (myeloid; both Pharmingen) combined with anti-HuCD45-allophycocyanin (APC) (Becton Dickinson) was performed. For all samples 7-amino actinomycin D (7-AAD; Sigma) was included to gate out dead cells. A minimum of 50 000 BM cells were examined for each sample. Only mice with both positive myeloid and lymphoid engraftment (defined as > 10 positive events each per 50 000 viable BM cells) were evaluated as positive. Gates were set so that samples incubated with irrelevant isotype-matched control Abs, had a maximum of 1 positive event per 50 000 BM cells analyzed. If no engraftment was detected by flow cytometry or if the myeloid engraftment was questionable, BM cells were plated in methylcellulose supplemented with human-specific cytokines (rhGM-CSF 50 ng/mL, rhIL-3 25 ng/mL, rhSCF 25 ng/mL) and rhEpo 5 U/mL at a density of 105 cells/35-mm plate, 4 replicates per group. Granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies were scored after 10 to 12 days. No colonies were observed in the absence of cytokines or from BM of untransplanted mouse cultured with the same cytokines (I.D. and S.E.W.J., unpublished observation, 2000).Long-term culture-initiating cell assay Long-term cultures were established and maintained according to previously described procedures.39 Briefly, stroma cell feeders were established by seeding a mixture (1:1) of 2 irradiated (8000 cGy) murine fibroblast cell lines (M2-10B4 and sl/sl, kindly provided by D. E. Hogge; Vancouver, BC, Canada), engineered to produce high levels of human G-CSF, IL-3, and KL,39 into 96-wells-collagen-coated microtiter plates containing long-term culture (LTC) medium (Myelocult; Stemcell Technologies) supplemented with freshly dissolved 106 M hydrocortisone
21-hemisuccinate (Sigma). The cytokine production from M2-10B4 and
sl/sl fibroblasts was tested (by commercially available enzyme-linked
immunosorbent assay [ELISA] kits; R & D Systems) and found to be at
recommended levels39 (I.D. and S.E.W.J., unpublished observation).
Freshly isolated CD34+CB cells (150 cells/well) or sorted
CD34+CD38 Flow cytometric evaluation of apoptosis, cell-cycle status, and differentiation of cultured CB progenitors Apoptosis was assessed by measuring redistribution of phosphatidylserine using annexin V-PE (Pharmingen) and uptake of 7-AAD.40 Briefly, CD34+ CB cells cultured for 6 days in KFT3 in the absence or presence of TNF were washed and resuspended in 100 µL annexin V binding buffer (Pharmingen) and incubated with anti-CD34-APC, annexin V-PE (5 µL), and 7-AAD (5 µg/mL) for 15 minutes, resuspended in annexin V-binding buffer and analyzed on a FACSCalibur.The CD34+ CB cells cultured for 6 to 7 days in the absence or presence of TNF were also subjected to high-resolution cell-cycle analysis as recently described41,42 with minor modifications. Fixation and permeabilization of cells were performed with Cytfix/Cytoperm Kit (Pharmingen). After washing, cells were stained with FITC-conjugated anti-Ki-67 (Immunotech, Westbrook, ME) or an isotype-matched irrelevant control Ab. After 3 hours of incubation in phosphate-buffered saline (PBS) containing 5% FCS supplemented with 7-AAD (5 µg/mL) on ice, samples were analyzed on a FACSCalibur. To evaluate differentiation of in vitro cultured (KFT3 in the absence or presence of TNF 20 ng/mL; 2-12 days) CD34+ CB cells were stained with anti-CD34-APC (Becton Dickinson) and a lineage cocktail containing PE- or FITC-conjugated Abs (CD3, CD14, CD15, CD19, CD20, CD56, CD66b, glycophorin A), and 7-AAD to exclude nonviable cells. Control samples of cultured cells were stained with irrelevant isotype-matched control Abs. Samples were analyzed on a FACSCalibur.
CD34+CD38 CB cells were cultured in KFT3 for
2 days, TNF had no effect on cell numbers (I.D. and S.E.W.J.,
unpublished observation, 2000), whereas following 5 to 8 days
of incubation in KFT3, by which time virtually all cells have
proliferated,23 KFT3 + TNF cultures contained 29%
(CD34+ cells), 67% (CD34+CD38
cells), and for CD34+CD38 BM cells 65% fewer
cells than KFT3 cultures (Figure 2).
However, cultures containing TNF expanded many-fold (Figure
2).
Next, single cell experiments were performed to establish whether
stimulation with TNF would block the first cell divisions of
CD34+CD38
TNF negatively affects maintenance of multipotent NOD-SCID repopulating HSCs The recent development and improvement of an assay that allows evaluation of human HSCs capable of in vivo reconstituting both myelopoiesis and lymphopoiesis in NOD-SCID mice has facilitated studies of candidate human HSCs and their regulation.31,35 When CD34+ or CD34+CD38 cells were
expanded in KFT or KFT3 under SF conditions, virtually all cells were
recruited into proliferation23 (Figure 3). This combined
with the fact that these conditions sustained NOD-SCID repopulating
cells (SRCs) capable of reconstituting human B and myeloid cells (Table
1 and Figure 3), suggested that SRCs had undergone cell divisions with sustained HSC function. In a total of 3 experiments, 9 of 9 mice transplanted with the EEs of 50 000 to
85 000 CB CD34+ cells or 5000 CD34+CD38 cells cultured for 5 to 8 days in
KFT3 showed human myeloid and B-cell reconstitution, with an average of
as much as 25% human engraftment. In contrast, only 1 of 9 mice
transplanted with the same EEs of cells cultured for 5 days in KFT3 in
the presence of TNF showed human myeloid and lymphoid engraftment
(1.1%; Table 1 and Figure 5). In
contrast, after 2 days of culture, at which time most candidate human
HSCs have not yet proliferated43,44 TNF did not affect
SRCs (Table 1). In a fifth experiment TNF was neutralized following 5 days of ex vivo expansion, and cells were cultured for an additional 3 days (Table 2). Although enhancing proliferation, the neutralization of TNF did not reverse the negative effect of TNF on SRCs, suggesting that the effect of TNF is
irreversible.
To further investigate whether TNF primarily targets actively
cycling HSCs, CD34+ cells were cultured in the presence of
Tpo alone, which promotes survival rather than proliferation of
candidate HSCs.45 Under such conditions the addition of
TNF did not adversely affect SRCs following as much as 5 days of
culture (Table 3).
Maintenance of cycling CB and adult BM
CD34+CD38 CB cells (Figure
6A,B) or
CD34+CD38 BM cells (Figure
7) in KFT3. Furthermore, as observed in
the in vivo reconstitution experiments, TNF at both 2 and 20 ng/mL, reduced the number of LTC-CFCs by more than 98%.
To exclude that the effect of TNF on LTC-ICs was indirect, an
additional experiment was performed in which
CD34+CD38 Because it was possible that the negative effect of TNF on cycling HSCs
was dependent on the specific cytokines and in vitro conditions used,
we also examined the effect of TNF on human HSCs in vivo. Because HSCs
cycle rapidly on transplantation, NOD-SCID mice were transplanted with
CD34+ CB cells and treated with TNF or PBS (control) 24, 64, and 96 hours after transplantation. Although the short-term
reconstitution with human CD34+ cells was not affected by
treatment with TNF (Figure 8), the number
of LTC-CFCs recovered from the BM was dramatically reduced (Table
4). Thus, TNF negatively regulates in
vitro as well as in vivo maintenance of cycling HSCs.
The ability of TNF to negatively regulate human HSC maintenance is signaled through TNFR55 and is not dependent on the Fas pathway The biologic effects of TNF are mediated through 2 members of the TNF receptor superfamily, TNFR55 and TNFR75.46 To be able to dissect the relative role of these 2 receptors in regulating maintenance of candidate human HSC, mutant TNF molecules selectively binding to TNFR55 and TNFR75 were used.36 Whereas the mutant-binding TNFR55 mimicked the severe negative effect of TNF on LTC-CFC activity of ex vivo expanded CB and BM CD34+CD38 cells, the mutant targeting TNFR75
had little or no effect (Figure 9).
Because TNF has been demonstrated to up-regulate Fas expression
on hematopoietic progenitor cells,47,48 it has thus been speculated that the negative effect of TNF on hematopoiesis might be
mediated through the Fas pathway. Thus, TNF-containing cultures of
CD34+CD38
Effects of TNF on apoptosis, cell-cycle distribution, and differentiation of hematopoietic progenitor cells To address the potential mechanism, by which TNF might negatively affect HSC maintenance, we first examined whether TNF enhanced apoptosis of cultured progenitor cells, because the TNFR55 contains the death domain involved in apoptosis.49 Toward this aim CD34+ CB cells were cultured in KFT3 in the absence or presence of TNF (20 ng/mL) for 6 days and subsequently stained with annexin V-PE and 7-AAD, as previously described.40 Using this method "early apoptotic" cells appear as annexin V positive and 7-AAD negative, whereas "late apoptotic" cells appear as positive for both annexin V and 7-AAD. As expected,1,25 a significant fraction of cultured cells were apoptotic, but no difference was observed between cells cultured in the absence or presence of TNF (Figure 11). Also, when only remaining CD34+ cells were investigated, no enhanced apoptosis was observed in response to TNF.
Next, the cell-cycle status of cultured progenitors was investigated
with a combined 7-AAD and Ki67 staining, as previously described.41,42 In agreement with the efficient induction
of proliferation, CD34+ cells cultured in KFT3 for 6 to 7 days were all in G1 or in cycle as illustrated through Ki67
expression in all cells. However, TNF did not affect the cell-cycle
distribution of cultured cells (Figure
12).
Finally, the potential effect of TNF on progenitor cell differentiation
was investigated. Following 2 days of incubation, TNF had little or no
effect on CD34 or lineage expression of KFT3-cultured cells, whereas
following 5 days of incubation in KFT3, TNF down-regulated CD34
expression, which was accompanied by enhanced lineage differentiation (Figure 13). Strikingly, following 12 days of culture in the presence of KFT3 and TNF no CD34+
cells remained, and enhanced myeloid differentiation was observed (Figure 13). In contrast, a high percentage of cells cultured in KFT3
in the absence of TNF remained CD34+Lin
Tumor necrosis factor has been implicated to be involved in BM failure in aplastic anemia and graft-versus-host disease (GVHD).5-7 TNF has also been demonstrated to inhibit the in vitro growth of human hematopoietic progenitors, including primitive progenitors.10,13 However, whether targets for TNF-induced growth inhibition include HSCs, functionally defined by their ability to reconstitute myelopoiesis and lymphopoiesis, have not been investigated, but is of considerable interest, because HSCs are sufficient and required for long-term hematopoietic reconstitution.1 In the present studies we demonstrate that TNF activation of the TNFR55
negatively affects maintenance of in vitro and in vivo cycling human
HSCs. Through recent studies we demonstrated that all murine and
candidate human HSCs are induced to undergo proliferation in vitro in
response to KL + FL + Tpo in the absence or presence of IL-3
under SF conditions.23,26 More importantly, such HSC
divisions were associated with sustained HSC function. Thus, this
represents a useful model for studying the in vitro regulation of HSC
self-renewal promoted by cytokines demonstrated to also be crucial for
in vivo HSC maintenance.2,3,18,19 The relevance of such a
model is also supported by studies showing similar kinetics of HSC
proliferation in vitro as in vivo following HSC
transplantation.50 Using this system we here demonstrate through single cell experiments that all candidate
CD34+CD38 The negative effect of TNF on HSCs appears to be primarily on cycling HSCs, because no effect of TNF was observed on SRCs when cells were cultured in Tpo alone, a condition demonstrated to promote survival rather than cycling of HSCs.45 Although we cannot exclude that the negative effect of TNF could be a consequence of compromised HSC engraftment or homing rather than an effect on self-renewal, it appears unlikely because in vitro LTC-ICs were compromised as much as in vivo SRCs. In addition, the negative effect of TNF on SRCs could not be reversed by neutralization of TNF and subsequent expansion, at least ruling out a reversible (cell-cycle-related) engraftment defect. In studies such as the present one, HSCs will always represent a minority of the cells investigated. Thus, an HSC-specific effect on cell cycle or apoptosis of TNF in the present studies would have been difficult to detect. In addition, when comparing data from single cell and bulk experiments, the role of indirect effects could differ and affect the results. However, in the present studies, the negative effects of TNF observed in these 2 systems appeared comparable. Thus, although the mechanism(s) responsible for the negative effect of TNF on HSC maintenance remains to be established in more detail, the present studies provide some important clues. First, we found no evidence for enhanced apoptosis in the presence of TNF following 6 days of culture, at which time TNF had abrogated virtually all HSC reconstituting activity. This is noteworthy because suppressive effects of TNF are frequently associated with enhanced apoptosis, mediated through the death domain of TNFR55.49 However, unlike the Fas receptor, TNFR55 also signals through alternative nonapoptotic pathways.49 TNF also did not affect cell-cycle distribution of cultured progenitors, but rather facilitated differentiation, as illustrated through down-regulation of CD34 and acquisition of lineage expression. Thus, the present data provide support for the intriguing hypothesis that TNF might regulate HSC fate through promoting their differentiation rather than self-renewal. Whether or not TNF plays a physiologic role in regulating the HSC pool remains unclear. Studies in TNFR55-deficient mice have revealed an increased number of phenotypically defined HSC,12 whereas other studies have suggested that such an increase does not necessarily translate into enhanced HSC function.52 In contrast there is ample evidence implicating involvement of TNF in the hematopoietic suppression observed in GVHD and bone marrow failure syndromes.5-7 The present findings make it possible that the targets for TNF-induced BM suppression might include HSCs, although further studies will be needed to address this.
We thank Dr Donna E. Hogge for kindly providing the murine fibroblast stroma cell lines and helpful advice regarding the use of these. We are in particular grateful to Dr Connie J. Eaves and Dr John E. Dick for helpful advice regarding the establishment and use of the LTC-IC and NOD-SCID assays. The expert assistance of Dr Lars Nilsson in the NOD-SCID assay, Gunilla Gärdebring, Lilian Wittman, and Ingbritt Åstrand-Grundström, as well as cell-sorting assistance by Carl-Magnus Högerkorp, Sverker Segrén, and Zhi Ma are highly appreciated. We also thank the staff and donors at the Department of Gynecology, Lund University Hospital and Helsingborg Hospital for help with providing CB, all volunteers for their BM contributions, and the physicians and other staff at the Department of Hematology, Lund University Hospital for performing the BM aspirations. The contributions by Dr Yutaka Sasaki, Dr Helga Björgvinsdóttir, and Dr Ewa Sitnicka through fruitful discussions and critical review of the manuscript are highly appreciated.
Submitted October 24, 2000; accepted May 18, 2001.
I.D. is supported through a fellowship from the Norwegian Cancer Society and the Faculty of Medicine, Norwegian University of Science and Technology. These studies were generously supported by grants from the Berta Kamrad Foundation; the Crafoord Foundation; the Georg Danielsson Foundation; the Gunnar, Arvid and Elisabeth Nilsson Foundation; the John and Augusta Persson Foundation; the O. and E. and Edla Johansson Foundation; the Thelma Zoega's Foundation; the Tobias Foundation; the Greta and Johan Kock's Foundations; the Royal Physiographic Society in Lund; ALF (Government Public Health Grant); Skånes Landsting; the Swedish Foundation for Strategic Research; the Swedish Cancer Society; Swedish Society of Pediatric Cancer and the Medical Faculty, University of Lund.
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.
Reprints: Sten Eirik W. Jacobsen, Department of Stem Cell Biology, Institute of Laboratory Medicine, University Hospital of Lund, Lund, Sweden; e-mail: sten.jacobsen{at}stemcell.lu.se.
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Top
Abstract
Introduction
(TNF) has been suggested to be
involved in a number of bone marrow failure syndromes.
-scintillation counter.
TNF) or 5 µg TNF (+TNF) 24, 64, and 96 hours after
transplantation. At 24 hours after the last injection (day 5) mice were
killed, and BM analyzed for human (HuCD45/71) and murine (MuCD45.1)
engraftment (as described in "Materials and methods"). Cells
positive for HuCD45/71 were analyzed for percentage HuCD34+
cells, which were sorted on a FACSVantage for subsequent studies in
LTC-IC assay (Table 
the role of TNF-alpha.
Transplantation.
1991;52:406-411