High Efficiency Recovery of Hematopoietic Progenitors from Peripheral Blood Harvests after Short- and Long-Term Cryopreservation.

Abstract Introduction: Transplantation of functional hematopoietic stem cells (HSC) using peripheral blood (PB), bone marrow (BM) or cord blood (CB) cells is widely used to treat malignant and nonmalignant disorders. Because long-term cryopreservation is performed for PB, BM and CB cells, and these are often used years after cell harvests, the implementation of a quality-assurance is a major requirement to ensure graft safety for clinical use. Methods: We assessed the efficiency of recovery of viable HSC from 37 patients (pts; n=20 NHL, n=6 Hodgkin, n=9 MM, n=2 AML) and 6 allogeneic-donors (AD) with stored PBSC samples. All pts had received an auto-PBSCT between 1992–2004. Stored PBSC samples used in this analysis had been cryopreserved for a median of 5.6 years (y; range: 1.3–12). We determined post-thawing recovery, cell viability, ex vivo expansion potential, CD34+ numbers, CFU growth in methylcellulose culture and LTC-ICs. Viable cells were determined by trypan blue and propidium iodide via FACS analysis, CFUs in 0.9% methylcellulose (supplemented with IMDM, 30% FCS and EPO, IL-3+GM-CSF) and LTC-IC as previously described. Pts and AD were analyzed as a total group and within 3 subgroups of: A) ‘long-term’ cryopreservation: n=21 PBSC harvests had a median cryopreservation of 9.5y (8–12), B) ‘short-term’ cryopreservation: n=16 harvests had a 2.9y (1.3–5.6) cryopreservation period, and C) n=6 pts showing delayed engraftment (EG) or early death after auto-PBSCT: the cryopreservation in these 6 pts was 2.7y (2.2–3.5). Cryopreservation results were correlated with clinical results and EG. Results: Hematopoietic EG in group A and B was prompt with WBC>1000/μl and platelets>20,000/μl on d10–11 post PBSC reinfusion. EG in group C was delayed albeit 4.3x106 CD34+ cells/kg bw (2.1–8.6) had been retransfused (WBC>1000/μl + platelets>20,000/μl: d+13 post PBSC infusion, non-platelet-EG >20,000/μl before death: n=5). Primary cause of death in group C was progressive disease in 3 and serious infections in 5 pts. Group A showed 74.3% viable cells post-thawing in PBSC grafts. Median number of CD34+ cells were 2.9%. Median numbers of CFU-C, BFU-E and GEMM were 36, 60 and 7, respectively. This was comparable with results in group B, showing 70% viable cells post-thawing, CD34+ cells of 4.2% and CFUs of 43, 75 and 6, respectively (p>0.05). Proliferative capacity was intact in both groups after 7 days of suspension culture, generating CFU-C, BFU-E and GEMM of 67, 29 and 1, respectively. In group C, viable cells were present in only 58% and median CFU-C, BFU-E and GEMM were 21, 5 and 0, respectively (p<0.05). After 7 days of suspension culture, total CFUs were 5 (<5% as compared to group A+B). Mean CFU-Cs before and after LTC-IC were 9 and 8 after LTC-IC culture in group C, whereas these were 18 and 16 in group A (p<0.05). Thus, the percentage of viable cells, CFUs and LTC-ICs was preserved after long-term cryopreservation (group A), showed no significant difference between group A+B, but were decreased in group C. Conclusions: We show that human PBSC can be stored for more than a decade without apparent loss of HSC activity and can be efficiently retrieved. These results reinforce that expiration dates cannot be set for safely stored cryopreserved HSC. Assessment of CD34+ cell numbers, clonogenic potential via methylcellulose and LTC-IC assays are clinically relevant, since they may correlate with clinical outcome. Thus, these hematopoietic assays are valuable to assess the quality of cryopreservation and possibly also outcome of PBSCT.

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Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2020-138529 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Sydney Felker ◽

Archana Shrestha ◽

Punam Malik

Keyword(s):

Lentiviral Vector ◽

Genetic Manipulation ◽

Ex Vivo ◽

Hematopoietic Progenitor Cell ◽

Homing Behavior ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference

Gene therapy/editing of CD34+ HSPC ex vivo, followed by their transplantation, can cure a variety of hematologic diseases. However, a substantial loss of HSPC occurs from collection to transplant. Losses occur during processing for HSPC enrichment, ex vivo genetic manipulation and culture, formulation, and testing prior to transplant. Further, HSPC are lost to peripheral organs during homing when delivered intravenously (IV), reducing the effective gm HSPC dose; a loss compounded by the lack of helper cells that aid in the homing and engraftment process which are removed during enrichment. Direct BM delivery of gm HSPC can overcome some of these limitations. This has been tried previously, with non-enriched whole cord blood (CB) and non-gm HSPC, with conflicting results. We hypothesized that BM delivery of a limited dose of gm adult HSPC would improve long-term repopulation over that of IV delivery by bypassing HSPC loss during homing. Using bioluminescent imaging, we determined that CB HSPC transduced with a luciferase lentiviral vector (LV) delivered by intra-femoral (IF) injection localized to the injected femur, validating our injection method. Next, we delivered mobilized peripheral blood (MPB) HSPC transduced with a GFP LV into irradiated NOD.LtSz-scid IL2rg -/- (NSG) mice via IV or IF injection in limiting dilution. Total human engraftment (hCD45+ cells), transduced human engraftment (hCD45+GFP+ cells), and multi-lineage engraftment were measured in the BM at 3- and 6-months post-transplant. HSPC gave rise to a bi-lineage (B-myeloid) graft at 3 months, suggesting hematopoietic progenitor cell (HPC) engraftment, and a multi-lineage graft (hCD33+, hCD19+, hCD3+, and hCD34+ cells) at 6 months, suggesting engraftment from a long-term repopulating cell or hematopoietic stem cell (HSC). At 3 months, IF delivery of HSPC resulted in significantly higher total and transduced human cell engraftment, measured in the non-injected femur (Table 1). The engraftment was bi-lineage. At 6 months, IF delivery of HSPC no longer significantly increased engraftment over IV delivery (Table 1). However, a multi-lineage graft was present, indicating full hematopoietic repopulation. There was no significant difference in the lineage output between either delivery method at 3 or 6 months. These data suggest that HPC homed and engrafted more efficiently than HSC, when delivered IF. Alternatively, IF delivery altered the BM microenvironment, allowing preferential homing of HPC. However, CD34- cells injected IF, to simulate pressure and passage of cells through the BM with IF delivery, followed by IV delivery of CD34+ cells (sham IF with IV HSPC delivery) resulted in similar homing patterns to CD34+ cells delivered IV (p=0.1, Figure 1A), suggesting that differences between IV and IF delivery were likely due to cell-intrinsic rather than cell-extrinsic differences between HPC and HSC. To study the mechanism of preferential engraftment of HPC over HSC with IF delivery, we analyzed expression of the major homing receptors CXCR4 and VLA-4 on HPC and HSC. CXCR4 (Figure 1B) and VLA-4 were both expressed at significantly higher levels on HPC than on HSC (CXCR4 p<0.01; VLA-4 p<0.05) and their expression increased with increasing culture time and with HSPC cycling. However, VLA-4 expression was significantly increased in GFP+ (MFI 65313 ± 4750) compared to GFP- (MFI 48969 ± 2099; p<0.01) HSPC. CXCR4 expression was similar in both GFP+ (MFI 4261 ± 189) and GFP- (MFI 5245 ± 1186) HSPC, mimicking the in vivo engraftment pattern of GFP+ and GFP- cells, suggesting that CXCR4 may be the molecule responsible for enhancing HPC homing and engraftment with BM delivery. An initial experiment shows that when we remove the high CXCR4 expressing CD34+38+ HPC and deliver HSC-enriched CD34+38- cells IV or IF, IF delivery results in higher long-term engraftment (additional experiments ongoing, Figure 1C, D). These data support the hypothesis that cell-intrinsic differences in the homing behavior of HSC and HPC is likely due to their differential expression of CXCR4. Studies underway on blockade of CXCR4 or VLA-4 on gm HPC and/or gm HSC followed by their IF or IV delivery will be presented. Overall, we show IV delivery of gm HSPC is comparable to BM delivery. However, as HSC-enriched cells become clinically available for genetic therapies, BM delivery of enriched gm HSC may result in superior engraftment. Disclosures Malik: Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

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

Blood ◽

10.1182/blood.v130.suppl_1.709.709 ◽

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.

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Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽

10.1182/blood.v91.4.1243 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1243-1255 ◽

Cited By ~ 69

Author(s):

Mo A. Dao ◽

Ami J. Shah ◽

Gay M. Crooks ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Low Levels ◽

Ex Vivo Culture ◽

Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

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Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽

10.3390/cells9040811 ◽

2020 ◽

Vol 9 (4) ◽

pp. 811

Author(s):

Pranav Oberoi ◽

Kathrina Kamenjarin ◽

Jose Francisco Villena Ossa ◽

Barbara Uherek ◽

Halvard Bönig ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

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Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽

10.1182/blood.v108.11.1329.1329 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1329-1329

Author(s):

Aleksandra Rizo ◽

Edo Vellenga ◽

Gerald de Haan ◽

Jan Jacob Schuringa

Keyword(s):

Cord Blood ◽

Cell Expansion ◽

Cultured Cells ◽

Ex Vivo ◽

Human Cord Blood ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

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Ex Vivo Expansion and Long-Term Hematopoietic Reconstitution Ability of Sorted CD34+CD59+ Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽

10.1182/blood.v112.11.2324.2324 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2324-2324

Author(s):

Juan Xiao ◽

Bing Han ◽

Wanling Sun ◽

Yuping Zhong ◽

Yongji Wu

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Peripheral Blood Cell ◽

Intravascular Hemolysis ◽

Blood Cell Count ◽

Normal Cells ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, venous thrombosis, and bone marrow (BM) failure. Until now, allogeneic hematopoietic stem cell transplantation is still the only way to cure PNH. Eculizumab, although very promising, is not the eradication of the disease because of raising the possibility of severe intravascular hemolysis if therapy is interrupted. Here we enriched the residual bone marrow normal progenitor cells (marked by CD34+CD59+) from PNH patients, tried to find an effective way of expanding the progenitors cells used for autologous bone marrow transplantation (ABMT). Objective To expand CD34+CD59+ cells isolated from patients with PNH and observe the long-term hemaotopoietic reconstruction ability of the expanded cells both ex vivo and in vivo. Methods CD34+CD59+ cells from 13 patients with PNH and CD34+ cells from 11 normal controls were separated from the bone marrow monouclear cells first by immunomagnetic microbead and then by flow cytometry autoclone sorting. The selected cells were then cultivated under different conditions for two weeks to find out the optimal expansion factors. The long-term hematopoietic supporting ability of expanded CD34+CD59+ cells was evaluated by long-term culture in semi-solid medium in vitro and long-term engraftment in irradiated severe combined immunodeficiency(SCID) mice in vivo. Results The best combination of hematopoietic growth factors for ex vivo expansion was SCF+IL-3+IL-6+FL+Tpo+Epo, and the most suitable time for harvest was on day 7. Although the CD34+CD59+ PNH cells had impaired ex vivo increase compared with normal CD34+ cells (the biggest expansion was 23.49±3.52 fold in CD34+CD59+ PNH cells and 38.82±4.32 fold in CD34+ normal cells, P<0.01 ), they remained strong colony-forming capacity even after expansion ( no difference was noticed in CFCs or LTC-IC of PNH CD34+CD59+ cells before and after expansion, P>0.05). According to the above data, 11/13(84.3%) patients with PNH can get enough CD34+CD59+cells for ABMT after expansion. The survival rate and human CD45 expression in different organs was similar between the irradiated SCID mice transplanted with expanded CD34+CD59+ PNH cells and those with normal CD34+ cells (P>0.05). The peripheral blood cell count recovered on day 90 in mice transplanted with PNH cells, which was compatible with those transplanted with normal cells (P>0.05). On secondary transplantation, the peripheral blood cell count returned to almost normal on day 30 in mice transplanted with either PNH cells or normal cells. Lower CD45 percentage was found in secondary transplantation compared with primary transplantation but no difference between mice transplanted with different cells. Conclusion Isolated CD34+CD59+ cells from patients with PNH can be effectively expanded ex vivo and can support lasting hematopoiesis both ex vivo and in vivo. These data provide a new potential way of managing PNH with ABMT.

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Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Blood ◽

10.1182/blood.v118.21.1920.1920 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1920-1920

Author(s):

Santosh Saraf ◽

Hiroto Araki ◽

Benjamin Petro ◽

Kazumi G Yoshinaga ◽

Simona Taioli ◽

...

Keyword(s):

Peripheral Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Self Renewal ◽

Transcript Levels ◽

Epigenetic Modifiers ◽

Cd34 Cells ◽

Mobilized Peripheral Blood

Abstract Abstract 1920 Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2'-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures: No relevant conflicts of interest to declare.

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Use of Plerixafor to Overcome Stem Cell Mobilization Failure: Long Term Follow up of Patients Proceeding to Transplant Using Plerixafor Mobilized Stem Cells

Blood ◽

10.1182/blood.v118.21.4390.4390 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4390-4390 ◽

Cited By ~ 1

Author(s):

Abhinav Deol ◽

Judith Abrams ◽

Ashiq Masood ◽

Zaid Al-Kadhimi ◽

Muneer H. Abidi ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Side Effects ◽

Cell Count ◽

Peripheral Blood ◽

Cd 34 ◽

Cd34 Cells ◽

Significant Difference

Abstract Abstract 4390 Background: Plerixafor is a CXCR 4 antagonist which is now approved for use for stem cell (SC) mobilization with granulocyte colony stimulating factor (GCSF) in patients with non Hodgkin lymphoma (NHL) or multiple myeloma (MM). Prior to the approval of plerixafor, we enrolled 49 patients in a compassionate use protocol at our institution to mobilize SC for patients who previously failed at least one mobilization attempt. Methods: Patients received 0.24 mg/kg of plerixafor subcutaneously 9 –11 hrs prior to apheresis in addition to twice daily GCSF. Results: Median age of the patients was 64 years (range, 23–74 years). NHL was the most common diagnosis in 27 (55%) patients, followed by MM with 17(35%) patients and HD with 5 (10%) patients. Thirty nine patients (80%) had been treated with more than 2 chemotherapeutic regimens prior to the first attempt at stem cell collection. Thirty seven patients (76%) failed one previous mobilization attempt, while 12 (24%) had failed 2 or more previous attempts. Using the combination of Plerixafor and GCSF we collected ≥ 2.5 × 106 CD34+ cells/Kg in 33 patients (67%). The median days for pheresis were 1 day with a range of 1 to 3 days. The median SC dose collected was 4 × 106 CD34+ cells/Kg, with a range 2.5 – 14.3. The median CD-34+ peripheral blood count on the 1st day of their collection with plerixafor was 22.4/uL. In contrast the median peripheral blood CD-34+ cell count in these patients on the day of their first collection which failed was 6.2 /uL. The median increase using G-CSF and plerixafor was 14.9 CD-34+ cells/uL. We collected ≥ 2.5 × 106 CD34+ cells/Kg on 4/5 (80%) patients with HD, 13/17 (76%) patients with MM and 16/27 (59%) patients with NHL. Sixteen patients (33%) collected < 2.5 × 106 CD34+ cells/Kg. The median cell dose collected in these patients was 1.4 × 106 CD34+ cells/Kg with a range, 0.4–2.2. The median number of days of pheresis was 2 days (range, 1–4 days). In these16 patients the median CD-34+ count on the day of their previous failed collection was11.2/uL. Their CD-34+ cell count on their first day of collection after the use of G-CSF and plerixafor was 8.3/ul. Figure 1 shows the change in peripheral CD34 counts with the prior mobilization attempt and after plerixafor mobilization, for 38 patients in whom data was available. The most common side effects attributed to plerixafor were diarrhea, fatigue, thrombocytopenia and bone pain; observed in 12%, 8%, 8% and 6% patients, respectively. Forty three of the 49 patients proceeded to an autologus peripheral blood SC transplant, 34 patients received ≥ 2.5 × 106 CD34+ cells/Kg. Thirty two of these patients used the plerixafor collection as the only source of SC. Two patients had their plerixafor mobilized SC combined with a previous suboptimal SC collection. Nine patients received < 2.5 × 106 CD34 + cells/Kg; 4 patients received plerixafor mobilized SC alone, 5 patients received plerixafor mobilized SC combined with their previously mobilized SC. The preparative regimens used were R- BEAM (20 patients), Melphalan (16 patients), BEAM (6 patients) and Etoposide+TBI (1 patient). All patients received GCSF from day +6 till WBC engraftment. The median days of WBC and platelet engraftment were day +11 (range, 9–13 days) and day +16 (range, 11–77 days), respectively. There was no significant difference in days to engraftment between the patients who collected greater or less than 2.5 × 106 CD34 + cells/Kg. With a median follow up 13.7 months, long term engraftment data is available on 27 patients. The median white cell count, hemoglobin and platelet count 1 year after transplant was 4.7 × 109/L, 12.2 g/dL and 109 ×109/L, respectively. There was no significant difference in counts at the 1 year mark between patients who collected more or less than 2.5 × 106 CD34 + cells/Kg. To date 15 patients have evidence of disease progression. Two patients have developed MDS/AML post transplant. Conclusion: Overall, plerixafor leads to mobilization of sufficient stem cells in a vast majority of patients who have failed previous mobilization attempts and allows more patients to proceed to an autologous SC transplant. Plerixafor is well tolerated with minimal side effects, acceptable time to engraftment and acceptable peripheral blood counts at 1 yr after the transplant. Our analysis suggests that failure to increase peripheral CD34 count after plerixafor when compared to previous attempts may predict unsuccessful mobilization. Disclosures: Lum: Transtarget Inc: Equity Ownership, Founder of Transtarget.

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Conditioned Medium Represents a Useful Solution to Increase the Expansion of Multipotent Progenitors with Strong Platelet Engraftment Activity

Blood ◽

10.1182/blood.v126.23.4276.4276 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4276-4276

Author(s):

Ahmad Abu-Khader ◽

Gwendoline Bugnot ◽

Manal Alsheikh ◽

Roya Pasha ◽

Nicolas Pineault

Keyword(s):

Cell Expansion ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Background Level ◽

Erythroid Progenitors ◽

Cellular Mechanisms ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Cd34 Cells

Abstract Delayed neutrophil and platelet engraftment is a significant issue of cord blood (CB) transplantation. Ex vivo expansion of CB hematopoietic stem and progenitor cells (HSPC) before infusion has been shown to accelerate hematopoietic recovery in patients. Recently, we reported that serum free medium (SFM) conditioned with osteoblasts derived from human bone marrow (BM) mesenchymal stromal cells, referred as M-OST CM, was superior to SFM or MSC CM for the expansion of CB CD34+ cells, and that HSPC expanded in M-OST CM provided better platelet engraftment. Since large number of expanded cells were transplanted in the original study, it was not possible to estimate the increased expansion of HSPC with short-term (ST) and long-term (LT) thrombopoietic and BM engraftment activities. The objectives herein were to investigate these shortcomings using limit dilution analysis (LDA) in transplantation assay and to investigate the cellular mechanisms at play. M-OST CM was prepared by conditioning SFM with immature M-OST overnight. CB CD34+ cells were expanded in M-OST CM or in SFM (defined as control) for 7 days with SCF, FL and TPO. CB cell expansion was significantly greater in M-OST CM cultures vs. SFM control (2.4 ±0.9 fold, mean ± SD, n=4, p=0.01). LDA transplantation assays were done by infusing the progeny of 500-8000 CD34+ cells in NSG mice. First, we compared the ST (< 31 days) and LT (˃ 100 days) thrombopoietic activities of expanded HSPC by measuring circulating human platelets (hPLT). The threshold for hPLT engraftment was set above the mean background level measured in control mice + 1SD. The median ST levels of hPLT in M-OST mice tended to be greater (2.5-fold, p˃0.05) in M-OST recipients (21 mice/condition, n=2). The frequency of ST hPLT HSPC estimated by LDA was 3.4 ±0.2 fold higher in M-OST CM cultures though the difference vs. control was not significant (p=0.11). LT hPLT levels were significantly greater in M-OST recipients (median 33 vs. 8 hPLT/uL blood, p=0.0027). Consistent with this, the frequency of HSPC with LT hPLT engraftment was increased in M-OST CM cultures (3.5±0.8 fold, p<0.04). Considering the differences in cell expansion, the net expansion of HSPC with ST and LT hPLT engraftment were raised by 5.5 ±1.7 and 6.0 ±3.4 fold in M-OST CM cultures vs. control (n=2). Next, LT human BM engraftment was analyzed at week 16. Preliminary results (13 mice/condition) suggest that the frequency of LT Scid repopulating cells (SRC) was increased by 27% in the M-OST CM culture vs. SFM control (frequency of 1/2878 vs. 1/3626 of day 0 starting cell). Next, we set to determine how M-OST CM increases the thrombopoietic activity of expanded CB HSPC. First, cytometry analysis (CD34, CD38, CD45RA, CD90 and CD123) revealed that M-OST CM preferentially increased the expansion of common myeloid progenitors (CMP, 8-fold, p=0.2, n=3), megakaryocyte-erythroid progenitors (MEP, 7-fold, p=0.02) and granulocyte-macrophage progenitors (GMP, 9-fold, p=0.02) vs. SFM control. Expansion of HSC-enriched cells was unchanged while that of multipotent progenitors (MPP) was reduced 2-fold (p<0.05). We set to confirm these results by culturing purified primary CB HSPC subsets in M-OST CM or SFM; M-OST CM induced greater expansions of MEP (3-fold), GMP (˃10-fold) while expansion in MPP cultures was greater with SFM control (1.5-fold). No growth was noted with the HSC and CMP cultures likely due to low sort yields. To complement these findings, we measured the expansion of myeloid CFU progenitors and long term culture-initiating cells (LTC-IC) by LDA. The total number of CFU was increased 2.4-fold (<0.02, n=4) by M-OST CM due mostly to increased expansion of CFU-G/GM colonies (2-fold, p<0.05) and BFU-E (2-fold, p=0.05). M-OST CM also sustained a 3.4-fold increase in LTC-IC expansion vs. SFM culture, though this finding remains to be confirmed in ongoing experiments. Finally, we investigated the effect of M-OST CM on the chemotaxis of HSPC toward SDF-1 since we previously reported increased expression of its receptor CXCR4 on CB cells in M-OST CM cultures. M-OST CM HSPC showed a modest 15% increase in migration vs. SFM control (n=4, p=0.10). In conclusion, our results demonstrate that the ST and LT hPLT engraftment activities of ex vivo expanded CB HSPC can be increased 5-6 folds by the use of M-OST CM due to the expansion of immature CB HSPC subsets including perhaps LT SRC. Whether M-OST CM can also modulate the homing activity of HSPC remains unclear. Disclosures No relevant conflicts of interest to declare.

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Factors Influencing Long-Term Hematopoietic Function Following Autologous Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.2186.2186 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2186-2186

Author(s):

Alissa Visram ◽

Natasha Kekre ◽

Christopher N. Bredeson ◽

Jason Tay ◽

Lothar B. Huebsch ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Infection Rates ◽

Advisory Committees ◽

Cd34 Cells ◽

Significant Difference

Abstract Background/Objective: Mobilized peripheral blood hematopoietic progenitor cells are the most common stem cell source for autologous hematopoietic stem cell transplantation (auto-HSCT). Successful short-term stem cell engraftment requires collection of at least 2x106 CD34+ cells/kg. The American Society of Bone Marrow Transplantation (ASBMT) recommends a stem cell infusion target of 3-5 x106 cells/kg (Giralt et al. 2014). However, the number of CD34+ cells to reinfuse to ensure long-term engraftment has not been established. Plerixafor, a reversible CXCR4 antagonist, increases CD34+ cell yield at collection even in patients who are predicted poor mobilizers (PPM). Although plerixafor could be used universally for all collections, this may not be the most cost-effective strategy (Veltri et al. 2012). This study sought to determine the minimum number of CD34+ cells/kg required for adequate long-term hematopoiesis, identify factors associated with poor long-term hematopoiesis, and determine if plerixafor mobilization improved long-term peripheral blood counts. Methods: A retrospective chart review was conducted on patients who underwent auto-HSCT between January 2004 and September 2013 at The Ottawa Hospital, for management of hematological malignancies. Peripheral blood cell counts were collected from 1 to 5 years after auto-HSCT, or until disease relapse. Poor long-term hematopoiesis was defined as an ANC <1 x109/L, hemoglobin <100 g/L, or platelets <100 x109/L. Patients were stratified into groups based on the infused CD34+ concentration (in cells/kg), and the proportion of patients with poor long-term hematopoiesis at 1, 2, 3, 4, and 5 years post auto-HSCT was compared with chi square tests. Long-term clinical outcomes (platelet and packed red blood cell transfusions, and post auto-HSCT infection rates) were compared between plerixafor-mobilized patients and PPM (defined as patients with pre-collection CD34+ <2 x 106 cells/kg) with standard mobilization regimens. Results: This study included 560 patients who underwent auto-HSCT, 210 with multiple myeloma and 350 with lymphoma. At 1 and 5 years post auto-HSCT 377 and 104 patients were included, respectively. A dose dependent improvement 1 year after auto-HSCT was seen in patients who received 0-2.99 x 106 CD34+ cells/kg (24.4%, n= 41) compared to patients who received 5-9.99 x 106 CD34+ cells/kg (11%, n=154, p=0.051) and ³10 x 106 CD34+ cells/kg (4.5%, n=66, p=0.006). Though there was a trend towards lower CD34+ infusions and poorer hematopoietic function (see table 1), there was no statistically significant difference in hematopoietic function based on CD34+ infusion concentrations after 1 year post auto-HSCT. 10 patients received <2 x106 CD34+ cells/kg, of whom the rate of inadequate hematopoiesis was 33% at 1 year (n=6) and 0% (n=1) at 5 years post auto-HSCT. Factors that increased the risk of poor hematopoiesis over the course of study follow up, based on a univariate analysis, included advanced age (OR 1.189, p=0.05), multiple prior collections (OR 2.978, p=0.035), and prior treatment with more than two chemotherapy lines (OR 2.571, p=0.02). Plerixafor-mobilized patients (n=25), compared to PPM (n=197), had a significantly higher median CD34+ cell collection (4.048 x109/L and 2.996 x109/L cells/kg, respectively, p=0.005). There was no significant difference in overall cytopenias, transfusion requirements, or infection rates between plerixafor-mobilized and PPM patients over the course of the study follow up. Conclusion: Low pre-collection CD34+ counts, advanced age, multiple prior collections, and more than two prior chemotherapy treatments adversely affected long-term hematopoiesis post auto-HSCT. We support the transfusion target of 3-5 x 106 cells/kg, as proposed by the ASBMT, given that at 5 years post auto-HSCT there was no statistical or clinically significant difference in hematopoietic function with higher CD34+ infusion targets. While mobilization with plerixafor significantly increased overall CD34+ cell collection when compared with PPM, long-term hematopoietic function and clinical outcomes were not different. This finding supports the practise of limiting plerixafor use only to patients who are PPM, thereby facilitating adequate stem cell collection and early engraftment, as opposed to universal plerixafor mobilization. Disclosures Sabloff: Lundbeck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Alexion: Honoraria.

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