Hematopoietic Progenitor Cell Products Research Articles

Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Outcomes with allogeneic stem cell transplant using cryopreserved versus fresh hematopoietic progenitor cell products

BackgroundAllogeneic hematopoietic stem cell transplant (alloHSCT) is a mainstay of treatment for hematologic malignancies such as acute leukemias and aggressive lymphomas. Historically, fresh hematopoietic progenitor cell (HPC) products have been preferred to cryopreserved products (cryo-HPC) due to concerns of loss of stem cell viability and number with the cryopreservation procedure. ObjectiveWe aimed to analyze the outcomes of patients who received cryo-HPCs during the COVID-19 pandemic and compare this against historical cohorts that received fresh HPC. Study DesignA retrospective chart review was conducted on all adult patients who received a peripheral blood alloHSCT in British Columbia, Canada between June 2017 and November 2021. Baseline characteristics, Kaplan-Meier (KM) overall survival (OS), engraftment, and incidences of acute and chronic graft versus host disease were compared between patients who received cryo-HPCs and fresh HPCs. Univariable analysis followed by multivariable analysis was performed using a backward stepwise selection procedure to generate predictors of OS, cumulative incidence of relapse (CIR), nonrelapse mortality (NRM), and primary and secondary graft failure. ResultsThree hundred eighty-three patients were included in the analysis, with cryo-HPC representing 40%. Median viability was higher in the fresh-HPC group at 99.2% (IQR 98.3–99.5) versus cryo-HPCs at 97.0% (96.0, 98.6) (P < 0.01). The 12-month actuarial survivals were 77% in the fresh HPC and 75% in the cryo-HPC groups (P = 0.21). There were no differences between cryo-HPCs and fresh HPCs on univariable analysis of OS, CIR, or NRM. There was a shorter median time to platelet engraftment in patients receiving fresh HPC at 17 days (IQR 16, 20) versus cryo-HPC at 21 days (IQR 18, 29), P < 0.001. There was a shorter median time to neutrophil engraftment in the fresh HPC group at 17 days (IQR 14, 20) versus 20 days (17, 23), P < 0.001. Cryo-HPC accounted for 5 out of 6 cases of primary graft failure (P = 0.04), and 3 out of five cases of secondary graft failure (P = 0.39). There were no significant differences in acute GVHD between the fresh HPC and cryo-HPC groups (P = 0.34). The incidence of moderate or severe chronic GVHD was 32% in the fresh-HPC group and 17% in the cryo-HPC group (P < 0.001).In multivariable analysis, cryopreservation did not emerge as an independent predictor of OS, CIR, NRM, primary GF or secondary GF. However, viability <90% on arrival at our center was a significant predictor of OS (HR 5.3, 2.3–12.3, P < 0.01), primary graft failure (OR 36.3, 5.4–210.2, P < 0.01), and secondary graft failure (OR 18.4, 1.7–121.1, P < 0.01). ConclusionsPatients who received cryo-HPCs had similar OS and relapse rates to those who received fresh-HPCs but typically took 2–3 days longer to achieve engraftment of platelets or neutrophils and were associated increased primary graft failure. However, after accounting for multiple variables, cryopreservation was no longer a significant predictor of survival or engraftment while viability <90% emerged as an important predictor of OS, primary graft failure, and secondary graft failure. If confirmed, this suggests that viability on arrival at the infusion center may be a good quality control indicator used to identify HPC products that may warrant recollection if the risk of graft failure is sufficiently increased.

Trem-Cel, a CRISPR/Cas9 Gene-Edited Allograft Lacking CD33, Shows Rapid Primary Engraftment with CD33-Negative Hematopoiesis in Patients with High-Risk Acute Myeloid Leukemia (AML) and Avoids Hematopoietic Toxicity during Gemtuzumab Ozogamicin (GO) Maintenance Post-Hematopoietic Cell Transplant (HCT)

Background: Allogeneic HCT is the standard of care for eligible patients with AML harboring high-risk features such as adverse-risk cytogenetics or measurable residual disease (MRD), however, the majority of these patients still relapse post-HCT and have very poor outcomes. The use of maintenance therapies intended to control post-HCT relapse is often limited by hematopoietic toxicity towards normal engrafted cells. GO (tradename: Mylotarg TM) is an anti-CD33 antibody-drug conjugate targeting CD33+ AML, but its use is limited by on-target, off-tumor hematopoietic toxicity toward CD33+ myeloid and progenitor cells. Tremtelectogene empogeditemcel (trem-cel; formerly VOR33) is a hematopoietic stem and progenitor cell product, manufactured from CD34+ cells isolated from a patient-matched donor, that has been modified by CRISPR/Cas9 gene-editing to lack CD33. Trem-cel was developed to protect normal hematopoietic cells from post-HCT CD33-directed therapies and allow exclusive targeting of residual leukemia. Methods: VBP101(NCT04849910) is a first-in-human Phase 1/2 open-label multicenter trial to establish the safety of using trem-cel as a donor allograft for CD33+ AML patients who are at high risk of relapse and undergoing myeloablative HCT followed by treatment with GO. Eligible patients (18-70 y) must have CD33+ AML with high-risk features for relapse, such as adverse-risk cytogenetics or MRD, and a 10/10 human leukocyte antigen (HLA)-matched related or unrelated donor. Donors undergo mobilization with G-CSF and plerixafor prior to apheresis. Trem-cel is manufactured from isolated CD34+ cells and cryopreserved. Patients undergo either a busulfan- or TBI-based myeloablative conditioning regimen prior to transplantation with trem-cel. After engraftment and recovery of a sufficient CD33-negative neutrophil count, patients begin maintenance therapy with GO in a 3+3 dose escalation strategy starting at 0.5 mg/m 2 every 28d for 4-8 cycles. Results: To date, 6 patients between 32-68 y (median 63.5 y) have been treated with trem-cel at a median dose of 5.2 x 10 6 CD34+ cells/kg (2.6 - 7.6) and CD33 editing efficiency of 88% (80 - 91). Primary neutrophil engraftment occurred in all patients after a median of 10 days (9 - 11) and platelet recovery occurred after a median of 16 days (15-22) excluding one patient with documented anti-platelet antibody (Fig 1). At the day 28 assessment, full peripheral blood (PB) myeloid chimerism was achieved in all patients and CD33 expression by flow was absent in a median of 94% of neutrophils (86-99) and 92% of monocytes (82 - 94), respectively. Comparable CD33 edits were detected in donor cells across myeloid and lymphoid lineages. GO dosing is ongoing at 0.5 mg/m 2 dose and neutrophil and platelet counts remain stable, suggesting protection from GO-mediated hematotoxicity. Following the 0.5 mg/ m 2 dose, the exposures of GO were predictively higher in the context of CD33-negative hematopoiesis relative to GO doses of 1-2 mg/m 2 (C max) and 4-5 mg/m 2 (AUC), respectively, in CD33+ relapsed/refractory AML patients. During GO dosing, the percent of CD33-negative cells increased in the PB and bone marrow (BM) across cell lineages consistent with enrichment of edited donor cells. Patients are followed for safety, hematologic protection from GO, disease status, and persistence of CD33 editing.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination

Background aimsCold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3–4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. Case reportsFour cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. ConclusionsWhile uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

Notch activation during early mesoderm induction modulates emergence ofthe T/NK cell lineage from human iPSCs.

A robust method of producing mature Tcells from iPSCs is needed to realize their therapeutic potential. NOTCH1 is known to be required for the production of hematopoietic progenitor cells with Tcell potential invivo. Here we identify a critical window during mesodermal differentiation when Notch activation robustly improves access to definitive hematopoietic progenitors with T/NK cell lineage potential. Low-density progenitors on either OP9-hDLL4 feeder cells or hDLL4-coated plates favored Tcell maturation into TCRab+CD3+CD8+ cells that express expected Tcell markers, upregulate activation markers, and proliferate in response to Tcell stimulus. Single-cell RNAseq shows Notch activation yields a 6-fold increase in multi-potent hematopoietic progenitors that follow a developmental trajectory toward Tcells with clear similarity to post-natal human thymocytes. We conclude that early mesodermal Notch activation during hematopoietic differentiation is a missing stimulus with broad implications for producing hematopoietic progenitors with definitive characteristics.

Trial in Progress: A First-in-Human Phase 1/2 Study of Hematopoietic Stem Cell Transplantation (HCT) with VOR33 (CD33-Deleted Allograft) Followed By Treatment with Gemtuzumab Ozogamicin (GO) in Patients with CD33+ Acute Myeloid Leukemia (AML) Who Are at High-Risk of Post-HCT Relapse

Background: Allogeneic HCT is the standard of care for eligible patients with AML harboring high-risk features such as adverse-risk cytogenetics or minimal residual disease (MRD). However, the majority of such patients still relapse post-HCT and have very poor outcomes. The use of maintenance therapies intended to control post-HCT relapse is often limited by hematopoietic toxicity towards normal engrafted cells. Gemtuzumab ozogamicin (GO, tradename: Mylotarg) is an FDA-approved anti-CD33 antibody-drug conjugate targeting CD33+ AML, but its use is limited by on-target hematopoietic toxicity towards CD33+ myeloid and progenitor cells. VOR33 is a CRISPR/Cas9 gene-edited allogeneic hematopoietic stem and progenitor cell (HSPC) product lacking CD33 protein that was developed to protect normal hematopoietic cells while exclusively targeting leukemia with CD33-directed therapies. VOR33 is manufactured from CD34+ cells isolated from healthy human leukocyte antigen (HLA)-matched donors. Pre-clinical murine and primate transplant studies using CD33-deleted HSPCs demonstrated unaltered long-term engraftment, multi-lineage hematopoietic differentiation and function, as well as long-term persistence of CD33 gene-edited hematopoietic cells. Additionally, subsequent treatment with GO eradicated CD33+ leukemia cells while the normal hematopoietic cells that reconstituted from CD33-edited HSCs were preserved. The improved therapeutic index for GO in combination with VOR33 has the potential to allow effective targeting of residual leukemia in AML patients post-HCT without dose-limiting hematopoietic toxicity. Study Design and Methods: VBP101(NCT04849910) is a first-in-human Phase 1/2 open-label multicenter trial to establish the safety of using VOR33 as a donor allograft for CD33+ AML patients who are at high risk of relapse and undergoing myeloablative HCT followed by treatment with low-dose GO. Participants (18-70 y) must have a diagnosis of CD33+ AML, an HLA-matched related or unrelated (10/10) donor and be a candidate for myeloablative HCT. Additional key inclusion criteria include CR1/CRi/CR2 with intermediate/poor cytogenetics and the presence of MRD. Patients with 5-10% bone marrow-only blasts with any cytogenetic risk are also eligible. Key exclusion criteria include prior auto/allo-HCT or prior treatment with GO. Related and unrelated donors undergo mobilization with G-CSF and plerixafor prior to cell collection. Manufacturing of VOR33 and isolation of an unmanipulated backup graft are performed centrally and both are cryopreserved. Subjects undergo either a busulfan- or TBI-based myeloablative conditioning regimen prior to transplantation with VOR33. After engraftment and recovery of a sufficient CD33-negative neutrophil count (~Day 60), patients begin maintenance therapy with GO. Part 1 of the study will enroll 9-18 evaluable patients to be treated in a 3+3 dose-escalation strategy with GO (0.5-2.0 mg/m2) in a 28-day cycle for up to 4 cycles. Part 1 will evaluate the safety of VOR33/GO and will determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of GO. The Part 2 expansion phase will enroll an additional 12 patients to further evaluate the safety of VOR33 and the preliminary efficacy of VOR33 and GO at the RP2D. A separate cohort is included for early relapse with treatment per the investigator's choice and may include use of GO. The primary endpoint is to assess the safety of VOR33 as measured by successful neutrophil engraftment at 28 days post-HCT. Secondary endpoints include time to neutrophil and platelet engraftment, assessment of the overall safety of VOR33 and GO (GvHD, graft failure, transplant-related mortality, RP2D, and MTD), non-hematological GO-related toxicities, GO pharmacokinetics, the efficacy of VOR33 in conferring protection from GO-related hematological toxicities, percent CD33 negative myeloid cells over time and survival rates (RFS and OS). Exploratory endpoints include the persistence of VOR33 gene editing and immune reconstitution. Study follow-up is 2 y with up to 15 y long term follow-up in a rollover safety study, VBP201 (NCT05309733). A Dose Escalation Committee and Data Safety Monitoring Committee will review GO dosing levels and monitor overall trial safety, respectively. VBP101 is currently open for enrollment and recruiting patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment of Individuals with Severe Sickle Cell Disease with OTQ923, an Autologous, Ex Vivo, CRISPR/Cas9-Edited, CD34+ Hematopoietic Stem and Progenitor Cell Product, Leads to Durable Engraftment and Fetal Hemoglobin Induction

Introduction: Increased levels of fetal hemoglobin (HbF; encoded by γ-globin [HBG1/HBG2]) are protective against complications of sickle cell disease (SCD). BCL11A is a transcription factor that binds to HBG1/HBG2 promoters and produces a γ-to-β globin (fetal-to-adult hemoglobin) switch. Inhibiting BCL11A binding to its target can reverse the switch and induce HbF expression in adult red blood cells (RBCs). OTQ923 is an autologous, ex vivo, CRISPR/Cas9-edited, CD34+ cellular product with a targeted disruption of the HBG1/HBG2 promoters on chromosome 11. Targeting this region in preclinical models led to HbF induction, phenotypically recapitulating hereditary persistence of fetal hemoglobin (a naturally occurring condition whose co-inheritance ameliorates SCD severity). This approach is more targeted than complete elimination of BCL11A expression in erythrocyte precursors for treating hemoglobinopathies. Additionally, unlike lentiviral vector gene addition, induction of endogenous γ-globin transcription could concur with reduction of βS-globin expression to avoid α- and β-like globin chain imbalance. Methods: Subjects with severe SCD, defined by a history of stroke, recurrent vaso-occlusive events (VOE), acute chest syndrome (ACS), chronic transfusions, recurrent priapism or red cell alloimmunization, were eligible to participate in the adult cohort (≥18-≤40 years old) of this clinical trial (NCT04443907). OTQ923 was manufactured from cryopreserved, peripheral blood derived CD34+ cells, collected after plerixafor mobilization. Prior to infusion, participants received myeloablative busulfan. Primary endpoints are to evaluate engraftment, long-term safety, and efficacy of HbF expression. Results: As of the data cut-off (July 8, 2022), 2 participants had received OTQ923, with follow-ups of 9 months (mos) and 3 mos, respectively. Here, we present initial clinical data from these 2 participants; additional data will be presented at the meeting. Both participants were homozygous for the sickle mutation. Participant 1 is a 22-year-old male with a history of silent cerebral infarcts, ACS, and priapism. At study entry, he was receiving chronic transfusions. He received 2.8x106 CD34+ cells/kg of OTQ923 (2 combined manufacturing batches). Participant 2 is a 21-year-old male with a history of ACS, and a silent cerebral infarct and was receiving hydroxyurea. He received 5.99x106 CD34+ cells/kg of OTQ923 (3 combined manufacturing batches). Time to neutrophil engraftment (the first of 3 consecutive days of absolute neutrophil count above 500 cells/mL after cell infusion) was 26 and 20 days for Participants 1 and 2, respectively. No OTQ923-related adverse events (AEs) were reported in either participant. All observed AEs were consistent with busulfan conditioning. Participants received RBC transfusions around conditioning and have received none after engraftment. For Participant 1, HbF level increased in the 6 mos after infusion and was stable at data cut-off (22.1% HbF at 9 mos after infusion). Induction of HbF was pancellular, with an F-cell percentage around 91% at 6 mos after infusion (Fig. 1A). Participant 2 had achieved an HbF level of 15.9% at 3 mos after infusion, the latest evaluable timepoint at data cut-off. Neither participant had any SCD-related VOEs after infusion of the cellular product. The proportion of edited alleles in the peripheral blood of Participant 1 was maintained at over 60% until Mo 6, which was the last evaluable timepoint (Fig. 1B). The indels were diverse, except for a predominant, large 5 kb deletion arising from simultaneous double-stranded DNA cleavage at HBG1 and HBG2 on-target sites, similar to previous reports. Conclusion: Preliminary data demonstrate clinically meaningful increases in HbF and evidence of benefit for both Participants 1 and 2 after OTQ923 infusion. Edited alleles were stable and maintained in the peripheral blood for >6 mos. The safety profile of OTQ923 was as expected after myeloablative busulfan and autologous hematopoietic stem cell transplantation. Taken together, these data suggest that this approach is safe and offers a potentially curative option for patients with severe SCD. Using cryopreserved, CD34+ cells as a starting material offers the advantage of centralizing manufacturing, scaling up the process to allow for improved patient access around the globe. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Handling of Allogeneic HPC Grafts in European Transplant Centers during the COVID-19 Pandemic - a Survey from the Infectious Diseases and Cellular Therapy & Immunobiology Working Parties of the EBMT

Background: For more than two years, the COVID-19 pandemic has had detrimental impact on medical practices. Because of its intrinsic complexity, the delivery of allogeneic hematopoietic cell transplantation (HCT) in this context represents an enormous challenge. Transplant centers have adapted promptly and developed strategies to enable safe HCT, by introducing significant changes in their procedures, e.g. through changes in donor types and an unprecedented use of cryopreservation for allogeneic hematopoietic progenitor cell (HPC) grafts. Methods: To explore how centers modified their strategies in terms of donor selection and implementation of new procedures (e.g. SARS-CoV-2 test strategy, cryopreservation), a 14-item survey was developed by the Infectious Disease (IDWP) & Cellular Therapy & Immunobiology (CTIWP) working parties of the European Society for Blood and Marrow Transplantation (EBMT). The survey was sent to 509 EBMT-affiliated allogeneic HCT in Q2, 2011 and re-opened between January 31st and March 18th, 2022 to also cover changes during to the infection period with the Omicron variant. Results: One hundred thirty-five of 509 (27%) centers from 31countries responded. Seventy-four percent of centers introduced COVID-19 mitigation measures for their donors (e.g. physical and social distancing) to minimize the risk of infection. Thirty-four percent of centers changed their donor search strategy, and favored recruitment of related haploidentical donors over unrelated donors (URD). In addition, 33% and 50% of centers searched for a backup donor in the related and URD settings, respectively. Only 10 (7.4 %) of centers resorted to cord blood (CB) as an alternative source to URD. Of these, four centers where pediatric centers and three, pediatric and adult transplant centers. Before COVID-19, only ten of 135 centers (7.4%) were routinely cryopreserving HPC products, whereas this percentage increased to 90% (122/135) during the pandemic. In details, 84/122 (69%) of centers cryopreserved both related and URD grafts, 34 (28%) only URD and 4 (3%) related grafts only. One hundred twenty centers responded during the re-opening of the survey to cover the situation during the Omicron wave. Of those, 78% were still considering graft cryopreservation, whereas 18/26 (70%) of centers not using cryopreservation during the Omicron wave used it before that period. In the majority of centers, changes in policies were introduced based on EBMT recommendations (88/117; 75%) and/or national guidelines (73/117; 62%). Bone marrow (BM) grafts were avoided by 52 (39%) centers (possibly because of the notorious low recovery percentage of cryopreserved BM progenitor cells). In the study period, a total of 4,443 related and 4,586 URD transplants have been reported. Of these transplants, 62% (n=2,748) related and 71% (n=3,254) of URD grafts have been cryopreserved pre-emptively and infused in the vast majority of cases. Sixty-four (2.3 %) related and hundred-eight (3.3 %) URD cryopreserved products have not been infused during the observation period. Release criteria for cryopreserved products in terms of COVID-19 were based on a negative SARS-CoV-2 test (not further specified) in 58/121 (48%), interview of the donor (medical history) in 4/121 (3%), and both in 38/121 (31%) of centers. Eighty-eight centers reported on their test strategy for related donors (multiple answers possible). A SARS-CoV-2 test was performed during check-up in 43% (38/88), before HPC mobilization in 63% (55/88) and before HPC collection in 41% (36/88) centers. Test strategies for URD were not covered by the survey. Conclusions: These data are limited by the low number of respondents, but show a historical increase in cryopreservation of allogeneic HPC grafts during the first three waves of the SARS-CoV-2 pandemic, more in URD than in related donor grafts. The use of BM decreased, and CB grafts were not used as an alternative at most centers. A significant proportion of cryopreserved products (2.8%) were not infused. The reason for this is under investigation. The clinical impact of allogeneic cryopreserved HPCs has to be further analyzed in a larger cohort of patients.

Sepax-2 cell processing device: a study assessing reproducibility of concentrating thawed hematopoietic progenitor cells

BackgroundAutologous hematopoietic progenitor cell (HPC) transplantation is currently the standard of care for a fraction of patients with newly diagnosed myelomas and relapsed or refractory lymphomas. After high-dose chemotherapy, cryopreserved HPC are either infused directly after bedside thawing or washed and concentrated before infusion. We previously reported on the comparability of washing/concentrating HPC post-thaw vs. infusion without manipulation in terms of hematopoietic engraftment, yet settled for the prior favoring cell debris and DMSO removal. For almost two decades, automation of this critical step of washing/concentrating cells has been feasible. As part of continuous process verification, we aim to evaluate reproducibility of this procedure by assessing intra-batch and inter-batch variability upon concentration of thawed HPC products using the Sepax 2 S-100 cell separation system.MethodsAutologous HPC collected from the same patient were thawed and washed either in two batches processed within a 3-4 h interval and immediately infused on the same day (intra-batch, n = 45), or in two batches on different days (inter-batch, n = 49) for those patients requiring 2 or more high-dose chemotherapy cycles. Quality attributes assessed were CD34+ cell recovery, viability and CD45+ viability; CFU assay was only performed for allogeneic grafts.ResultsIntra-batch and inter-batch median CD34+ cell recovery was comparable (75% vs. 73% and 77% vs. 77%, respectively). Similarly, intra-batch and inter-batch median CD45+ cell viability was comparable (79% vs. 80% and 79% vs. 78%, respectively). Bland-Altman analysis describing agreement between batches per patient revealed a bias close to 0%. Additionally, lower HPC recoveries noted in batch 1 were noted as well in batch 2, regardless of the CD34+ cell dose before cryopreservation, both intra- and inter-batch, suggesting that the quality of the collected product plays an important role in downstream recovery. Intrinsic (high mature and immature granulocyte content) and extrinsic (delay between apheresis and cryopreservation) variables of the collected product resulted in a significantly lower CD45+ viability and CD34+ cell recovery upon thawing/washing.ConclusionsAutomated post-thaw HPC concentration provides reproducible cell recoveries and viabilities between different batches. Implications of this work go beyond HPC to concentrate cell suspension/products during manufacturing of cell and gene therapy products.

Validation of automated fluorescent-based technology for measuring total nucleated cell viability of hematopoietic progenitor cell products.

A reliable rapid method for measuring total nucleated cell (TNC) viability is essential for cell-based products manufacturing. The trypan blue (TB) exclusion method, commonly used to measure TNC viability of hematopoietic progenitor cell (HPC) products, is a subjective assay, typically uses a microscope, and includes a limited number of cells. The NucleoCounter NC-200 is an automated fluorescent-based cell counter that uses pre-calibrated cartridges with acridine orange and DAPI dyes to measure cell count and viability. This study describes the validation of the NC-200 for testing HPC's viability. Samples from 189 fresh and 60 cryopreserved HPC products were included. Fresh products were tested for viability after collection by both TB and NC-200. 7-aminoactinomycin D (7AAD) CD45+ cell viability results were obtained from a flow cytometry test. Cryopreserved products thawed specimens were tested for viability by both TB and NC-200. The NC-200 viability results were compared with the other methods. Acceptability criteria were defined as ≤10% difference between the NC-200 method and the other methods for at least 95% of the samples. Fresh products' mean viability difference between NC-200 and TB or 7AAD CD45+ method was 4.9% (95%CI 4.6-5.4) and 2.8% (95%CI 2.2-3.4), respectively. Thawed products' mean viability difference between NC-200 and TB was 3.0% (95%CI 0.4-5.6). The NC-200 automated fluorescent-based method can be used effectively to determine HPC's viability for both fresh and cryopreserved products. It can help eliminate human bias and provide consistent data and operational ease.

Integrins, anchors and signal transducers of hematopoietic stem cells during development and in adulthood.

Hematopoietic stem cells (HSCs), the apex of the hierarchically organized blood cell production system, are generated in the yolk sac, aorta-gonad-mesonephros region and placenta of the developing embryo. To maintain life-long hematopoiesis, HSCs emigrate from their site of origin and seed in distinct microenvironments, called niches, of fetal liver and bone marrow where they receive supportive signals for self-renewal, expansion and production of hematopoietic progenitor cells (HPCs), which in turn orchestrate the production of the hematopoietic effector cells. The interactions of hematopoietic stem and progenitor cells (HSPCs) with niche components are to a large part mediated by the integrin superfamily of adhesion molecules. Here, we summarize the current knowledge regarding the functional properties of integrins and their activators, Talin-1 and Kindlin-3, for HSPC generation, function and fate decisions during development and in adulthood. In addition, we discuss integrin-mediated mechanosensing for HSC-niche interactions, ex vivo protocols aimed at expanding HSCs for therapeutic use, and recent approaches targeting the integrin-mediated adhesion in leukemia-inducing HSCs in their protecting, malignant niches.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. V: Manufacture and quality control

Chimeric antigen receptor T cells (CAR-T), especially against CD19 marker, present in lymphomas and acute B leukemia, enabled a revolution in the treatment of hematologic neoplastic diseases. The manufacture of CAR-T cells requires the adoption of GMP-compatible methods and it demands the collection of mononuclear cells from the patient (or from the donor), generally through the apheresis procedure, T cell selection, activation, transduction and expansion ex vivo, and finally storage, usually cryopreserved, until the moment of their use. An important aspect is the quality control testing of the final product, for example, the characterization of its identity and purity, tests to detect any contamination by microorganisms (bacteria, fungi, and mycoplasma) and its potency. The product thawing and intravenous infusion do not differ much from what is established for the hematopoietic progenitor cell product. After infusion, it is important to check for the presence and concentration of CAR-T cells in the patient's peripheral blood, as well as to monitor their clinical impact, for instance, the occurrence of short-term, such as cytokine release syndrome and neurological complications, and long-term complications, which require patient follow-up for many years.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Volume determination methods for hematopoietic progenitor cell products collected by apheresis

CD34+ cell count of hematopoietic progenitor cell products is often first determined using a product sample, with the final reported cell count obtained by multiplying cell count from the sample by the total product volume. Product volume may be determined by apheresis instruments used for collection or calculated based on specific gravity (SG). Here the authors sought to determine the discrepancies between these methods and the impact on patient care. A total of 262 products collected from 176 donors by apheresis were retrospectively reviewed. Volumes calculated by apheresis instruments were compared with volumes calculated based on SG. CD34+ cell/kg doses based on volumes from apheresis instruments and SG were also compared. Furthermore, among 42 patients who required multiple collections, the authors determined whether different calculation methods would have changed the number of procedures required. The volumes for 253 products (96.6%) and CD34+ cell/kg doses for 257 products (98.1%) generated from apheresis instruments and SG were similar. Products with discrepant volumes when calculated using different methods were not associated with delayed engraftment. Forty patients who underwent multiple collections (95.2%) were under their collection goals during their first collection and would have required multiple procedures regardless of which method was used to calculate product volumes. Volumes determined based on SG did not provide a clear benefit over volumes determined by apheresis instruments. Hence, variation in volume calculation methods across different laboratories is likely to lead to minimal impact on patient care.

A Metabolic Interplay Coordinated By Hlx Balances Hematopoietic Stem Cell Maintenance and Differentiation

Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with acute myeloid leukemia, but its mechanism of action remains obscure. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased numbers of immature myeloid cells at 48 hpf (hours post fertilization), thereby mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX under control of either an endothelial or HSPC specific promoter. To delve into the molecular mechanism of HLX function, we performed expression analysis of sorted endothelial cells from zebrafish that overexpress HLX and hlx1 morphants. Overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (NDUFS4, SDHD, CYTB, COX7A2) and upregulation of the peroxisome proliferator activated receptor family member δ (PPARδ). Downregulation of hlx1 led to diminished expression of PPARδ. Analysis of open chromatin by ATAC-seq revealed altered chromatin accesibility around the vicinity of these genes in hlx1 morphants. To validate functionally these results we measured the mitochondrial membrane potential by Tetramethylrhodamine, Methyl Ester, Percholate (TMRM). TMRM was decreased upon HLX overexpression and increased in hlx1 morphants. These results suggested that HLX regulates the fate of hematopoietic cells by fine tuning mitochondrial membrane potential and PPARδ. Indeed PPARδ agonists could rescue HSPC generation in zebrafish morphants, while PPARδ antagonists relieved the differentiation block of myeloid cells. Direct regulation of mitochondrial genes and PPARδ by HLX was established by chromatin immunoprecipitation in human cell lines. To test whether there is a conserved, intrinsic role of HLX in primary hematopoietic progenitor cells, we used human progenitor CD34+ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased numbers of colonies in colony forming unit assays and a myeloid differentiation block, whereas downregulation greatly compromised the ability of CD34+ cells to form colonies. HLX overexpression resulted in diminished oxidative phosphorylation and mitochondrial membrane potential whereas downregulation of HLX coincided with increased mitochondrial membrane potential. PPARδ agonists and antagonists could rescue TMRM staining and the colony forming capacity in HLX knockdown CD34+ cells and alleviated the myeloid differentiation block upon HLX overexpression, respectively. In summary, our findings show that HLX balances hematopoietic stem and progenitor cell maintenance and differentiation by modulating their metabolic status. DisclosuresNo relevant conflicts of interest to declare.

Potentially modifiable predictors of cell collection efficiencies and product characteristics of allogeneic hematopoietic progenitor cell collections.

Hematopoietic progenitor cell (HPC) and immune effector cell (IEC) therapies often require high doses of mononuclear cells (MNCs), whether CD34+ cells, lymphocytes, or monocytes. Cells for IEC can be sourced from HPC products. We thus examined potentially modifiable variables affecting collection efficiencies (CEs) of MNC subsets in HPC collection and also of the typically undesired cell types of platelets, granulocytes, and red cells, which hinder downstream processing. Finally, we sought to confirm previously indeterminate studies of the effect of an adjusted collect flow rate (CFR) on CD34+ CE. We performed univariate and multivariate regression analyses of all 135 National Marrow Donor Program (NMDP) HPC collections in 2019 and compared these fixed CFR procedures to previous NMDP collections using adjusted CFRs. Target cell CEs decreased with increasing peripheral blood (PB) concentration and were associated with different cell type locations within the MNC layer. CEs of undesired cell types varied with standard procedural parameters (inlet flow rate, whole blood processed, etc.). Interestingly, some CEs increased with preapheresis hematocrit. Finally, adjusting the CFR by PB MNC count improved MNC CE but not CD34+ CE. Correlation of target cell CEs with their PB concentration and different cell type locations by depth within the MNC layer indicates the importance of investigating the compensatory fine-tuning of procedure variables to improve CE. Correlation of CEs with PB hematocrit, and CFR adjustment by a modified PB MNC and/or PB CD34 algorithm should be further explored. Adjusting standard procedural parameters may reduce product contamination.

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation

AbstractFanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.

Suppression of Hematopoietic Activity in Tenascin-C–Deficient Mice

AbstractTenascin-C (TN-C), a member of the extracellular matrix (ECM) glycoprotein family, is expressed on the surface of stromal cells in the hematopoietic system or lymphoid organs. Recently, TN-C–deficient mutant mice produced by TN-C gene targeting through homologous recombination were shown to develop normally, although TNs have been reported to play important roles in organogenesis and carcinogenesis. In the present study, we found that colony-forming capacity of bone marrow (BM) cells was considerably lower in TN-C–deficient mice (a decrease of ∼35% from control), although their mononuclear cell count and BM architecture showed no significant difference from those of normal mice. Furthermore, in long-term BM culture in vitro, hematopoietic cell production (a decrease of ∼40% in Dexter's condition and of ∼65% in Whitlock-Witte's condition from control), colony-forming capacity of the produced cells (a decrease of ∼60% from control), and longevity of the cultures were markedly lower in the TN-C–deficient mice than in control mice, whereas hematopoiesis in the TN-C–deficient mutant mice was sustained. The addition of TN-C glycoprotein to long-term BM cultures of TN-C–deficient mice clearly induced the recovery of hematopoietic cell production and colony-forming capacity of hematopoietic progenitor cells. Thus, these results provide direct evidence that an ECM glycoprotein component, TN-C, plays a relevant role in hematopoiesis through interactions between stromal cells and hematopoietic progenitor cells.