We previously reported the generation of pluripotent and ultracompetitive HSCs through modulation of Hoxb4 and Pbx1 levels. These Hoxb4hiPbx1lo HSCs display a tremendous regenerative potential, yet ...they are still fully responsive to in vivo regulatory signals that control stem cell pool size (20 000 HSCmouse) and differentiation pathways. Further work in our laboratory attempted to circumvent these physiological constraints by expanding Hoxb4hiPbx1lo transduced HSCs in vitro, and hence revealing their intrinsic expansion potential. Independent experiments were performed where primary mouse BM cells were co-infected with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP (double gene transfer ranged between 20–50%). Hoxb4hiPbx1lo HSCs measured using the CRU assay expanded by 105-fold during a 12 day in vitro culture. Following serial transplantations, these cells displayed an additional 4–5 log expansion in vivo. Total stem cell content per animal remained within normal limits. Southern blot analyses of proviral integrations showed that the expansion was polyclonal, and analyses of individually expanded clones provided a molecular proof of in vitro self-renewal (SR). This unprecedented level of HSC expansion in such a short time course (105-fold in 12 days) implies an absolute HSC doubling time of approximately 17 hours in our culture, raising the possibility that virtually all dividing HSCs undergo self-renewal. This analysis prompted us to dissect the impact of Hoxb4 on cell proliferation versus cell fate (SR?).
When analyzed during the period of maximal HSC expansion, the cell cycle distribution of Sca+ or Sca+Lin− cells were comparable between the cultures initiated with neo control versus Hoxb4 BM cells (CTL vs Hoxb4: G0/G1: 66% vs 83%; S: 15% vs 9%; G2/M: 18% vs 7%). Correspondingly, CFSE tracking studies confirmed the identical, or even lower, number of cellular divisions in Sca+ cells isolated from cultures initiated with Hoxb4 versus neo transduced cells. Annexin V studies precluded protection from apoptosis as the major mechanism to increase HSC numbers since similar results (3–10% positive cells) were observed in the Hoxb4 versus neo-transduced cells.
In summary, our studies support the emerging concept that distinct molecular pathways regulate cell proliferation and self-renewal, suggesting that Hoxb4 + antisense Pbx1 predominantly triggers self-renewal over HSC proliferation.
One of the major obstacles to the successful clinical application of hematopoietic stem cell (HSC) transplantation, particularly in the context of related haplotype-mismatched transplantation, ...unrelated cord blood transplants for adults, and grafts that are processed ex vivo to remove malignant, or alloreactive T cells, is the number of available long-term repopulating HSCs. The addition of soluble recombinant TAT-HOXB4 protein was recently reported to enable rapid in vitro expansion of murine HSCs that retain their in vivo proliferation and differentiation capacity. However, the ability of this recombinant protein to effectively expand human hematopoietic stem cells remains hypothetical. In addition, limited information is available on underlying mechanisms of HOXB4 HSC expansion. First, to determine the capacity of recombinant TAT-HOXB4 protein to promote human HSC expansion, we treated human CD34+ cells for 4 and 8 days with 40 nM, or 80 nM TAT-HOXB4 protein in X-Vivo 15 medium supplemented with Stem Cell Factor, TPO, IL-6 and Flt3-ligand. Cultures exposed to TAT-HOXB4 treatment for 8 days had no pronounced effect on the total cell yield. During this period, a 2-fold net loss of CFU-GEMM was observed for controls, in comparison to ~8-fold and ~5-fold expansions in response to 40 nM and 80 nM TAT-HOXB4 (p<0.05), respectively. Recombinant TAT-HOXB4 also induced ~10–15-fold expansion of large CFU-GM, compared to only ~2.5-fold increase for controls (p<0.05). HSC numbers were enumerated at the beginning and at the end of the treatment using the principle of limiting dilution in a 4-month NOD/SCID repopulation assay. Culture for 8 days in cytokines devoid of TAT-HOXB4 resulted in ~2-fold loss of SCID Repopulating Cells (SRCs), while cultures supplemented with 40 and 80 nM TAT-HOXB4 protein showed a 2.5-fold (95% CI 1.7 – 3.3 fold) and 5.5-fold (95% CI 3.6 – 7.4 fold) increase, respectively. Then, to determine whether the increase in HSC numbers resulted from HOXB4-enhanced proliferation of HSCs, we examined the cell cycle profile of control and HOXB4-treated cell populations using Hoechst 33342 and pyronin Y dyes. After 4-day treatment with 80 nM TAT-HOXB4, 44%±12% of CD34+CD38+ cells were in Go, compared to only 19%±6% of the controls (p<0.05). In contrast, similar proportions (89–91%) of quiescent CD34+CD38− cells were observed for both conditions. Tracking cell divisions using CFSE also showed that during this period, HOXB4-treated CD34+CD38+ cells underwent ~2 population doublings less than controls (p<0.05). In conclusion, short-term exposure of human CD34+ populations to recombinant TAT-HOXB4 protein has the potential to achieve clinically relevant HSC expansion levels. At the HSC level, these observations suggest that TAT-HOXB4 preferentially affects cell fate (self-renewal?) rather than cell proliferation.
Murine squamous cell carcinoma (SCCVII) cells were genetically engineered to produce marine granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF immunotherapy, based on the peritumoral ...injection of lethally irradiated GM-CSF-producing SCCVII cells, was examined as adjuvant to photodynamic therapy (PDT) treatment of this tumor. The GM-CSF immunotherapy administered three times in 48-h intervals, starting 2 days before the light treatment, substantially improved the curative effect of Photofrin-mediated PDT. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative-mediated PDT. The tumor-localized GM-CSF immunotherapy alone had no obvious effect on the growth of parental SCCVII tumors. This treatment did not significantly alter the differential peripheral WBC count and appeared not to affect tumor leukocyte infiltration. However, GM-CSF treatment did increase the cytotoxic activity of tumor-associated macrophages against SCCVII tumor cells. It appears, therefore, that tumor-localized immune stimulation by GM-CSF amplifies a PDT-induced antitumor immune reaction, which has a potentiating effect on tumor control.
By modulating levels of Pbx-1, a homeodomain protein and DNA binding cofactor of Hoxb4, it is possible to generate pluripotent, ultracompetitive in vivo repopulating Hoxb4hi and Pbx1lo hematopoietic ...stem cells (HSCs) (Immunity, 2003, Krosl et al). Despite the tremendous regenerative potential demonstrated by these cells, the total in vivo HSC pool in recipients remained within physiological limits (~20 000 HSC/mouse), implying that environmental factors (niche availability?) restricted their expansion. These studies thus implied that bypassing the in vivo constraints of niche availability by culturing the transduced HSCs in vitro might reveal the intrinsic expansion potential of these cells. To study this hypothesis, Hoxb4hiPbx1lo transduced HSCs were generated by co-infecting primary mouse bone marrow (BM) cells with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP. At the end of the co-culture with retroviral producers, double gene transfer (Hoxb4hiPbx1lo ) was ~20% as determined by flow cytometric analysis of GFP and YFP co-expression. These cells were then cultured in the presence of serum and cytokines for additional 12–16 days, and the numbers of total cells, clonogenic progenitors and HSCs were determined at regular intervals. To quantify the magnitude of the in vitro HSC expansion, CRU assays for determination of cells with long-term lympho-myeloid repopulation potential were performed after removal of BM cells from co-culture with retroviral producers, and then at various time points. The increase in HSC numbers over time in culture was calculated as the ratio between absolute numbers of Hoxb4hiPbx1lo HSCs at a given time point, and their numbers at initiation of culture. In our initial experiment, numbers of Hoxb4hiPbx1lo CRU increased from 1000 at day 0 to 1.2 x 107 at day 12, for a net 10 000-fold expansion. After transplantation into irradiated mice, these cells underwent an additional 360-fold in vivo expansion to regenerate HSC pools of recipients up to, but not above, normal levels. Southern blot analyses of proviral integrations in DNA isolated from sorted Mac-1+, B-220+ and CD4+CD8+ cells derived from primary and secondary recipients demonstrated that cultured Hoxb4hiPbx1lo HSCs retained their ability to differentiate into all hematopoietic lineages examined. To estimate the numbers of distinct Hoxb4hiPbx1lo HSCs in cultures, BM cells from primary recipients of 1 x 106 cells from a 16-day expansion culture were plated in methylcellulose, and the clonal origin of individual myeloid colonies was determined by Southern blot analysis. These experiments showed that several distinct HSC clones reconstituted each recipient, and that some clones reconstituted at least 2 mice, illustrating the polyclonal nature and self-renewal activity of the cultured HSCs. Together, our experiments show that after unprecedented levels of in vitro expansion, Hoxb4hiPbx1lo HSCs remained capable of reconstituting myeloid and lymphoid systems of primary and secondary recipients, and yet responsive to the in vivo regulatory mechanisms that limit total stem cell pool size, reflecting the interplay between autonomous and non-cell autonomous control of HSC self-renewal. Decreasing Pbx1 levels in Hoxb4 overexpressing HSCs could thus be used as a tool for studying mechanisms of self-renewal and replicative senescence, and may lead to clinically relevant protocols for HSC expansion.
Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of
Hoxb4
and its co-factor
Pbx1
in primary hematopoietic cells
...(Hoxb4
hi
Pbx1
lo
cells
)
, we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 10
5
-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of
Hoxb4
hi
Pbx1
lo
stem cells present at initiation of culture underwent self-renewal in vitro. In this setting,
Hoxb4
and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Sca1
+
Lin
−
cells when compared to controls. Q-PCR analyses further revealed a down regulation of
Cdkn1b
(
p27
Kip1
) and
Mxd1
(
Mad1
) transcript levels in
Hoxb4
hi
Pbx1
lo
primitive cells, accompanied by a more subtle increase in
c-myc
and reduction in
Ccnd3
(Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.
Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of Hoxb4 and its co-factor Pbxl in primary hematopoietic cells ...(Hoxb4hiPbxl(10) cells), we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 105-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of Hoxb4hiPbxl(10) stem cells present at initiation of culture underwent self-renewal in vitro. In this setting, Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Scal+Lin- cells when compared to controls. Q-PCR analyses further revealed a downregulation of Cdknlb (p27Kipl) and Mxdl (MadI) transcript levels in Hoxb4hiPbxl(l0) primitive cells, accompanied by a more subtle increase in c-myc and reduction in Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.
The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously ...exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.
Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of
Hoxb4 and its co-factor
Pbx1 in primary hematopoietic cells
...(Hoxb4
hiPbx1
lo
cells
), we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 10
5-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of
Hoxb4
hiPbx1
lo
stem cells present at initiation of culture underwent self-renewal in vitro. In this setting,
Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Sca1
+Lin
− cells when compared to controls. Q-PCR analyses further revealed a downregulation of
Cdkn1b (
p27
Kip1
) and
Mxd1 (
Mad1) transcript levels in
Hoxb4
hiPbx1
lo
primitive cells, accompanied by a more subtle increase in
c-myc and reduction in
Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.
Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of Hoxb4 and its co-factor Pbx1 in primary hematopoietic cells ...(Hoxb4hi Pbx1lo cells ) , we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 105 -fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of Hoxb4hi Pbx1lo stem cells present at initiation of culture underwent self-renewal in vitro. In this setting, Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Sca1+ Lin− cells when compared to controls. Q-PCR analyses further revealed a downregulation of Cdkn1b ( p27Kip1 ) and Mxd1 ( Mad1 ) transcript levels in Hoxb4hi Pbx1lo primitive cells, accompanied by a more subtle increase in c-myc and reduction in Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.
To analyze the molecular mechanisms by which erythropoietin (Epo) can
stimulate proliferation and differentiation of hemopoietic cells, I studied the
effects of quantitative and qualitative ...alterations in the expression of
erythropoietin receptors (EpoRs) in hemopoietic cell lines and in the primary
bone marrow cells using retrovirus mediated gene transfer to engineer high
level expresson of normal and mutant EpoRs in these cells, lnterleukin-3 (IL-3)-
dependent murine bone marrow derived Ba/F3 cells engineered to express
normal EpoR increased their levels of β-globin mRNA in response to Epo, and
this partial differentiation correlated with a marked Epo-induced growth delay,
indicating that the transduced EpoR was capable of inducing a distinct set of
intracellular events. The tyrosine kinase inhibitor genistein blocked both Epoinduced
β-globin mRNA accumulation and proliferation in this model system. In
contrast, inhibition of protein kinase C by Compound 3 suppressed only Epo-induced
differentiation without affecting proliferation, indicating that the
proliferative and differentiation functions of the EpoR can be uncoupled. Mutant
EpoRs lacking all intracellular tyrosines were compromised only in proliferative
signaling, implying that tyrosine phosphorylation of the EpoR itself is not
required for its differentiation function. With IL-3 and Epo costimulation, IL-3
signaling appeared to be dominant, since no increase in β-globin mRNA
occurred. Chimeric EpoRs comprising the extracellular domain of the EpoR and
the transmembrane and cytoplasmic region of IL-3-R-βIL-3 were capable of
inducing β-globin mRNA accumulation, suggesting the existence of a second
EpoR subunit responsible for differentiation or that the a subunit of the IL-3 R
prevents it. Arguing against the former, a truncated EpoR lacking an intracellular
domain possessed no biological activity. Chimeric EpoRs comprising the
extracellular domain of the EpoR and the transmembrane and intracellular
domains of the IL-3R a subunit were, however, capable of transmitting the Epo-induced
mitogenic signal but failed to stimulate accumulation of β-globin mRNA.
Moreover, coexpression of EpoR/IL-3Ra with EpoR/IL-3R β-IL-3 suppressed
β-globin mRNA accumulation, which implicated an active role for the IL-3-Ra
subunit in inhibiting EpoR-specific differentiating signals.
Epo also exhibited a marked effect on proliferation of EpoR-transduced
primary mouse bone marrow cells. Epo alone supported proliferation of EpoRtransduced
CFU-GM and CFU-GEMM in semi-solid and suspension cultures,
indicating that Epo was capable of replacing other cytokines normally required
for the in vitro proliferation of non-erythroid and multipotent clonogenic
progenitors. No Epo-induced proliferation of control cells could be detected in
cultures containing high numbers of irradiated EpoR-transduced cells,
indicating that Epo stimulated proliferation directly, through activation of the
transduced EpoR, and arguing against the possibility of Epo-induced secretion
of growth factor(s) within the population of the EpoR-transduced cells.
To study effects of ectopic EpoR expression on proliferation of stem cells
in vivo, EpoR- and neo-transduced bone marrow cells were transplanted into
lethally irradiated mice. Recipients of the EpoR-transduced bone marrow
developed within 6-14 weeks severe anemia, leukocytosis characterized by
accumulation of undifferentiated blasts, and had significantly increased
numbers of all clonogenic progenitor classes, consistent with development of
myeloproliferative disease. Bone marrow and spleen cells recovered from the
affected mice expressed high levels of surface EpoRs and proliferated in
response to Epo, but not in the absence of growth factors, supporting a link between the Epo-induced deregulation in proliferation of the EpoR transduced
stem cells and development of neoplasia.
Together, the data presented in this thesis provide evidence that EpoRs
may influence both proliferative and differentiative decisions of hemopoietic
cells subject to their ability to interact with different signalling intermediates.
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