scholarly journals Inhibition of HDAC5-Mediated p65 Deacetylation Enhances Human Cord Blood Hematopoietic Stem Cell Homing and Engraftment in NSG Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 884-884 ◽  
Author(s):  
Xinxin Huang ◽  
Bin Guo ◽  
Hal E. Broxmeyer
Keyword(s):  
Promoter Region ◽  
Fold Increase ◽  
Treated Group ◽  
Vehicle Control ◽  
Cxcr4 Expression ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cxcr4 Mrna ◽  
M344 Treatment

Abstract Stromal cell derived factor-1α (SDF-1α)/chemokine C-X-C receptor 4 (CXCR4) interactions play a crucial role in hematopoietic stem cells (HSC) trafficking and homing to the bone marrow (BM) environment. To identify new epigenetic regulators of CXCR4 receptor, we screened a chemical compound library of epigenetic enzyme inhibitors to evaluate their effects on membrane CXCR4 expression in CB CD34+ cells. We found that treatment with a couple of histone deacetylase (HDAC) inhibitors, including M344, strongly upregulated membrane CXCR4 expression. We also investigated the effect of M344 on membranal CXCR4 expression in a rigorously defined more primitive HSC cell population (CD34+CD38-CD45RA-CD49f+CD90+) and found that there was a 2.5 fold increase in the M344 treated group compared with vehicle control treated cells. Quantitative RTPCR also showed increased CXCR4 mRNA levels in M344-treated CD34+ cells compared with vehicle control (3.1 fold), indicating that this regulation occurs at the transcriptional level. We next evaluated the effect of M344 treatment on HSC chemotaxis in in vitro transwell migration assays. Both vehicle and M344 treated CB CD34+ cells showed significant migration to 50ng/mL SDF-1α, however, chemotaxis was 2.1 fold higher in M344 treated group. Enhanced migration to SDF-1α by M344 was also observed in the more primitive HSC population. Chemotaxis of CB CD34+ cells to SDF-1α was blocked by CXCR4 antagonist AMD3100, suggesting that the effect was mediated through the CXCR4 receptor. To directly evaluate in vivo homing, vehicle and M344 treated CB CD34+ cells were injected into sublethally irradiated NSG mice, and human cells homing to mouse BM were analyzed 24 hours after transplantation. Consistently, M344 treatment enhanced CB CD34+ cells homing by 2.3 fold in NSG mice. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and M344 treated CB CD34+ cells. Poisson distribution analysis revealed an SRC frequency of 1/3216 in vehicle control treated group and 1/746 in M344 treatment. We calculated the respective presence of 310.9 SRCs and 1340.5 SRCs in 1×106 cells from vehicle control and M344-treated cultures, so M344 treatment resulted in a 4.3 fold increase in the number of functionally detectable SRCs compared with vehicle control. Eighteen HDACs have been identified in humans and they are divided into four classes. The mechanisms regarding HDAC regulation of HSC homing and engraftment are largely unknown. Using shRNA to knockdown expression of individual HDACs in CB CD34+ cells, we surprisingly found that HDAC5 shRNA transfection resulted in upregulation of membrane CXCR4 expression. LMK235, a selective inhibitor of HDAC5, also increased membrane CXCR4 expression in CB CD34+ cells. In contrast, inhibition of other HDACs did not show any effect on membrane CXCR4 expression. Similar to M344, LMK235 treatment resulted in significantly higher CXCR4 mRNA, membrane CXCR4 expression in CB HSCs, enhanced migration to SDF-1α in chemotaxis assay, and higher number of cells homed to the BM in NSG mice. We next performed chromatin immunoprecipitation (ChIP) assays to examine the chromatin status at the CXCR4 promoter region. H3K9 acetylation levels were significant higher in LMK235-treated CB CD34+ cells compared with vehicle control, suggesting increased H3K9 levels at the CXCR4 promoter region contributes to increased CXCR4 transcription. To further explore the mechanisms underlying HDAC5 regulation of HSC homing, we tested a couple of signaling pathways and found that inhibition of the NF-κB signaling pathway by Andrographolide and Pyrrolidinedithiocarbamate Ammonium, suppressed M344 and LMK235-mediated CXCR4 upregulation on CB CD34+ cells. It has been reported that acetylation of NF-κB p65 subunit enhances its transcriptional activity. We examined the acetylation levels of p65 and found that LMK235 treatment resulted in increased levels of p65 acetylation in CB CD34+ cells, indicating p65 could be a downstream target of HDAC5. Consistently, using ChIP assay we detected increased levels of acetylated p65 binding to the CXCR4 promoter region in the LMK235-treated group. Taken together, our results reveal previously unknown specific epigenetic regulation of HSC homing and engraftment by HDAC5, which suggests a new translational strategy to enhance HSC transplantation. Disclosures Broxmeyer: CordUse: Other: SAB Member.

Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3870-3870 ◽  
Author(s):  
Hai Cheng ◽  
Patali S. Cheruku ◽  
Luigi Alvarado ◽  
Ayla Cash ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Signaling Pathways ◽  
Research Funding ◽  
Fold Increase ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Inflammatory Conditions ◽  
Cd34 Cells

Abstract Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag's evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. Figure Figure. Disclosures Cheng: Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

scholarly journals Nitric Oxide Promotes Human Hematopoietic Stem Cell Homing and Engraftment Via cGMP-Pkg Signaling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 807-807 ◽  
Author(s):  
Xinxin Huang ◽  
Bin Guo ◽  
Maegan L. Capitano ◽  
Hal E. Broxmeyer
Keyword(s):  
Guanylyl Cyclase ◽  
Pde5 Inhibitor ◽  
Soluble Guanylyl Cyclase ◽  
Treated Group ◽  
Vehicle Control ◽  
No Donor ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract The success of hematopoietic stem cell (HSC) transplantation relies on adequate homing and long-term engraftment of HSC into the bone marrow (BM). The free radical nitric oxide (NO) is a gaseous molecule that plays important roles in a variety of physiological regulations. NO can freely diffuse across cellular membranes and activate an enzyme, soluble guanylyl cyclase, to produce cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP binding activates cGMP-dependent protein kinase (PKG) and other proteins to regulate many biological processes. However, the roles of NO and cGMP in regulating HSC function still remain poorly understood. To explore the importance of NO signaling in HSC, we first evaluated the effects of NO on human cord blood (CB) HSC chemotaxis in an in vitro transwell migration assay. We found that treatment of human CB CD34+ cells for 16 hours with an NO synthesis inhibitor, L-NAME, did not affect chemotaxis towards CXCL12. However, treatment of human CB CD34+ cells 16 hours with NO donor compound sodium nitroprusside (SNP) resulted in 55% more migration toward CXCL12 compared to vehicle control. Enhanced chemotaxis by SNP was also observed in a more primitive HSC cell population (Lin-CD34+CD38-CD45RA-CD49f+CD90+, 71% more migration) suggesting NO donor treatment promote HSC migration. The other two NO donors, SNAP and NOC5 showed similar effects on promoting CD34+ cells and HSC migration. NO activates soluble guanylyl cyclase in target cells, so next we explored the relationship between HSC migration and soluble guanylyl cyclase. By treating human CB CD34+ cells with riociguat, a soluble guanylyl cyclase stimulator, we found that riociguat treatment also resulted in 78% more HSC migration toward CXCL12. The other soluble guanylyl cyclase activator BAY412272 showed a similar effect as riociguat by promoting HSC chemotaxis (68% more migration compared with vehicle control). Inside the cell cGMP is degraded by phosphodiesterase 5 (PDE5), so a PDE5 inhibitor would suppress cGMP breakdown and activate cGMP signaling. Consistently, we found that CD34+ cells with PDE5 inhibitor avanafil or sildenafil treatment showed increased HSC chemotaxis compared with vehicle control (57% and 62% more migration respectively). Next we used PKG inhibitor KT5823 to test whether PKG is involved in HSC migration and found that KT5823 totally blocked the effects of SNP, riociguat and avanafil on enhanced HSC migration, suggesting that NO promote HSC migration through cGMP-PKG signaling. To directly evaluate in vivo homing, vehicle, SNP or riociguat treated CB CD34+ cells were injected into sublethally irradiated NSG (NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz) mice, and human cell homing to mouse BM, as indicated by human CD45 percentage, was analyzed 24 hours after transplantation. Consistently, SNP or riociguat treatment resulted in a 2.1 fold and 2.3 fold increase of human cell homing in NSG mice respectively compared with vehicle control treatment. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and riociguat treated CB CD34+ cells. Engraftment of riociguat-treated CB CD34+ cells was significantly increased in primary NSG recipient mice compared with that of vehicle control treated group four months after transplantation (47.6% vs 23.7%). Both human myeloid and lymphoid chimerisms were also increased. Poisson distribution analysis revealed an SRC frequency of 1/2977 in vehicle control treated group and 1/512 in Riociguat treatment, resulting in the presence of 335.9 SRCs and 1953.1 SRCs in 1×106 cells from vehicle control and riociguat-treated cultures. We are currently performing RNA-seq and quantitative proteomic analysis in riociguat-treated CB CD34+ cells to reveal specific downstream targets in regulating HSC homing. Taken together, our study suggests that human HSC homing and engraftment can be enhanced by modulating the NO/cGMP signaling pathway. Some compounds tested in our study, such as SNP, riociguat, avanafil are FDA approved medications broadly used for myocardial infarction, pulmonary hypertension and erectile dysfunction. So utilization of these drugs in HSC transplantation should be practical. Our work offers a new and simple approach to bolster the effectiveness of HSC transplantation. Disclosures No relevant conflicts of interest to declare.

AMD3100 Combined with Standard Doses of G-Csf Leads to Rapid, Consistent Mobilization of Hematopoietic Progenitor Cells in Patients with Non-Hodgkin’s Lymphoma (NHL) and Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2847-2847 ◽  
Author(s):  
Patrick Stiff ◽  
Ivana Micallef ◽  
Philip McCarthy ◽  
Margarida Magalhaes-Silverman ◽  
Neal Flomenberg ◽  
...  
Keyword(s):  
Fold Increase ◽  
Selective Inhibition ◽  
Treated Group ◽  
Median Number ◽  
Salvage Chemotherapy ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Cd34 Cells ◽  
Before And After ◽  
And Migration

Abstract CXCR4, the chemokine receptor for stromal derived factor-1 (SDF-1) plays an important role in the homing and migration of hematopoietic stem cells. Selective inhibition of the binding of the CXCR4 receptor by the molecule AMD3100 leads to the mobilization of hematopoietic progenitors, including CD34+ cells into the circulation, even in the steady state. Preliminary studies have indicated that AMD3100 given 10–11 hours before each apheresis along with standard doses of G-CSF mobilizes more CD34+ cells per kilogram than does G-CSF alone. A Phase II study of this combination in patients with NHL and myeloma is underway. G-CSF at 10μg/kg/day for a duration of up to 9 days is administered with AMD3100 at 240 μg/kg/day starting on the evening of day 4 of G-CSF therapy, (10–11 hours prior to first and subsequent daily aphereses) until 5 x 106 CD34/kg are collected, or for a maximum of 5 days of a standard 3 blood volume apheresis. Both agents are administered subcutaneously. To date 20 patients (myeloma-6, NHL-14) have been enrolled and analyzed. Of these 20, 14 are considered to have been ‘heavily pre-treated’ using standard definitions (≥ 10 cycles of chemotherapy, platinum based salvage chemotherapy and/or radiation therapy to bone marrow sites). Blood CD34 assays (cells/μl) were performed before and after each AMD 3100 dose and on each apheresis product. After the first dose of AMD3100 there was a 2.6 fold increase in CD34/μl in blood (23 → 60 CD34+ cells/μl). The median number of apheresis performed was 2 (range 1–5); the median total CD34 collected for all 20 patients was 5.7 x 106/kg (range 2.32–14.58 x 106/kg). All patients had collections of &gt; 2.0 x106 CD34/kg, and in 12 of 20 the 5 x 106 CD34/kg cell dose goal was collected, including 8 of 14 in the heavily pre-treated group. In fact in 6, the 5 x 106 CD34/kg cell dose was collected in a single apheresis. The median CD34/kg cell dose collected for the 14 heavily pre-treated patients was 5.7 x 106/kg (2.32–6.48 x 106/kg). There were no serious AE’s related to the use of AMD3100. Transient GI toxicity (mostly diarrhea) occurred in 10 of 20 patients shortly after the injections of AMD3100, but only 3 required therapy. Engraftment data is available for the first 12 patients treated. Time to ANC &gt;500/μl and platelets &gt; 20,000/μl was 9 (range 8–10) and 12 (range 9–19) days respectively. This novel combination of AMD3100 and G-CSF appears to be effective in mobilizing large numbers of CD34+ cells, even in patients considered to be heavily pre-treated, and is an effective alternative to chemotherapy/cytokine mobilization. Compared to studies of G-CSF alone, this combination may reduce the number of apheresis procedures needed to collect an adequate graft for rapid hematopoietic engraftment. The study is ongoing with accrual to date of 40 patients.

scholarly journals Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 709-709
Author(s):  
Bin Guo ◽  
Xinxin Huang ◽  
Hal E. Broxmeyer
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Metabolic Reprogramming ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ppar Γ ◽  
Control Shrna ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.

Human CD34+Cells Mobilized by AMD3100 Demonstrate Enhanced NOD/SCID Repopulating Function Compared to CD34+ Cells Mobilized by Granulocyte Colony Stimulating Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1962-1962 ◽  
Author(s):  
David A. Hess ◽  
Louisa Wirthlin ◽  
Timothy P. Craft ◽  
Jesper Bonde ◽  
Ryan W. Lahey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Paired Comparison ◽  
Fold Increase ◽  
Human Cells ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3722-3722
Author(s):  
Li Ming Ong ◽  
Xiubo Fan ◽  
Pak Yan Chu ◽  
Florence Gay ◽  
Justina Ang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Insulin Growth Factor ◽  
Nsg Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

Decreased Expression of HOXB4 Gene in Aplastic Anemia; Regulatory Interaction with GATA-2,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3436-3436
Author(s):  
Tohru Fujiwara ◽  
Hisayuki Yokoyama ◽  
Yoko Okitsu ◽  
Mayumi Kamata ◽  
Shinichi Fujimaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Promoter Region ◽  
Expression Vector ◽  
Gene Promoter ◽  
K562 Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Gene Promoter Region ◽  
Chip Analysis

Abstract Abstract 3436 Background: Aplastic anemia (AA) is characterized by a reduced number of hematopoietic stem cells (HSCs). It has been proposed that immunological injury in HSCs leads to reduced numbers of stem cells in the bone marrow. In addition, expression of the critical regulator of hematopoiesis GATA-2 is decreased in CD34-positive cells in AA (Fujimaki et al. Br J Haematol 2001). Despite the compelling results described above, only limited information has emerged regarding intrinsic abnormalities of hematopoietic stem cells in AA. It has been demonstrated that HOXB4 induces HSC expansion ex vivo (Antonchuk et al. Cell 2002), and restoring HOXB4 protein in HSCs from bone marrow failure patients promotes HSC expansion (Tang et al. Br J Haematol 2009). In conjunction with the evidence that recent genome-wide analysis of GATA factor chromatin occupancy identified GATA-2 peak at HOXB4 promoter (Fujiwara et al. Mol Cell 2009), we hypothesized that GATA-2 directly regulates HOXB4 expression in HSCs, which might contribute to the pathogenesis of AA. Here, we investigated possible link between GATA-2 and HOXB4, and also tested if HOXB4 is deregulated in CD34-positive cells from patients with AA. Method: For GATA-2 overexpression, human GATA-2 coding sequence was cloned into pcDNA3.1 expression vector as well as MSCV retroviral expression vector (Clontech). For GATA-2 knockdown, siRNA specific for human GATA-2 was transfected into CD34-positive cells or K562 cells by Amaxa Nucleofector kit (Amaxa Inc.). For promoter assay, DNA fragment of the HOXB4 gene promoter region (up to −262 from 1st ATG) was cloned into pGL3-Basic (Promega), and the GATA deletion construct was subsequently created with QuickChange™Site-Directed Mutagenesis Kit (Stratagene). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using antibodies for GATA-2 (H-116, Santa Cruz). For analyzing clinical samples, informed consent was obtained in all cases and ethical considerations according to the declaration of Helsinki were followed. Results: To examine if GATA-2 and HOXB4 are functionally linked, we transfected a GATA-2 expression vector into K562 cells, and demonstrated that GATA-2 significantly upregulated endogeneous HOXB4 expression. Furthermore, siRNA-mediated GATA-2 knockdown in K562 cells significantly reduced HOXB4 expression, indicating that HOXB4 is a GATA-2 target gene. We overexpressed/reduced GATA-2 in cord blood-derived CD34+ cells, which also provided evidence for GATA-2 regulation of HOXB4 expression. Promoter analyses revealed that GATA sequence located at −160/-157 of the HOXB4 gene promoter region was required to confer luciferase activity in K562 cells. In vitro DNA binding studies and quantitative ChIP analysis revealed specific GATA-2 occupancy at a chromatin region containing this element. Finally, we demonstrated that HOXB4 gene expression was significantly decreased in CD34+ cells from patients with AA (n=10) compared to those with ITP (n=13). The expression levels of HOXB4 and GATA-2 also correlated in these populations (r=0.6573, p<0.01). Conclusion: Based on these findings, we propose that decreased expression of GATA-2 in hematopoietic stem cells of AA leads to reduced HOXB4 transcription, which may have an important role in the development and/or progression of the disease. Disclosures: No relevant conflicts of interest to declare.

Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 143-143 ◽  
Author(s):  
Saar Gill ◽  
Sarah K Tasian ◽  
Marco Ruella ◽  
Olga Shestova ◽  
Yong Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Research Funding ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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