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Apheresis is essential to conducting hematopoietic cell transplantation and genetically engineered cellular therapy procedures. Many patients and donors require central venous catheter (CVC) access for apheresis due to lack of adequate peripheral venous access. CVC placement has risks of associated complications and requires additional institutional resources and expertise. We present a single institutional experience with 78 patients, who underwent a total of 87 apheresis procedures after placement of a midline catheter. This included 49 individuals who collected cells for immune effector cell therapy. Apheresis through midline catheter was successful in 81 out of 87 procedures. There were no midline catheter associated thromboses or infections. Six patients required placement of a backup CVC and were able to undergo apheresis without delay. Our experience shows that use of midline catheter for apheresis is feasible and generalizable to most populations requiring cellular therapy, with the potential to decrease utilization of limited healthcare resources., (© 2025 Wiley Periodicals LLC.)

Hematopoietic cell transplantation (HCT) is an important therapy for many hematological malignancies as well as some non-malignant diseases. Post-transplant hematopoiesis is affected by multiple factors, and the mechanisms of delayed post-transplant hematopoiesis remain poorly understood. Patients undergoing HCT often suffer from significantly reduced food intake due to complications induced by preconditioning treatments. Here, we used a dietary restriction (DR) mouse model to study the effect of post-transplant dietary reduction on hematopoiesis and hematopoietic stem cells (HSCs). We found that post-transplant DR significantly inhibited both lymphopoiesis and myelopoiesis in the primary recipient mice. However, when bone marrow cells (BMCs) from the primary recipient mice were serially transplanted into secondary and tertiary recipient mice, the HSCs derived from the primary recipient mice, which were exposed to post-transplant DR, exhibited a much higher reconstitution capacity. Transplantation experiments with purified HSCs showed that post-transplant DR greatly inhibited hematopoietic stem cell (HSC) expansion. Additionally, post-transplant DR reshaped the gut microbiotas of the recipient mice, which inhibited inflammatory responses and thus may have contributed to maintaining HSC function. Our findings may have important implications for clinical work because reduced food intake and problems with digestion and absorption are common in patients undergoing HCT., Competing Interests: Declarations. Ethics Approval: The Animal Experimental Ethical Inspection of Nanchang Royo Biotech Co., Ltd. (RYEI20170513-1) approved all mouse experiments. Consent for Publication: This manuscript has been approved by all authors and is solely the work of the authors named. Consent for Participate: Not applicable. Conflict of Interest: The authors have no conflicts of interest to declare., (© 2024. The Author(s).)

Aging is a process accompanied by functional decline in tissues and organs with great social and medical consequences. Developing effective anti-aging strategies is of great significance. In this study, we demonstrated that transplantation of young hematopoietic stem cells (HSCs) into old mice can mitigate aging phenotypes, underscoring the crucial role of HSCs in the aging process. Through comprehensive molecular and functional analyses, we identified a subset of HSCs in aged mice that exhibit "younger" molecular profiles and functions, marked by low levels of CD150 expression. Mechanistically, CD150 low HSCs from old mice but not their CD150 high counterparts can effectively differentiate into downstream lineage cells. Notably, transplantation of old CD150 low HSCs attenuates aging phenotypes and prolongs lifespan of elderly mice compared to those transplanted with unselected or CD150 high HSCs. Importantly, reducing the dysfunctional CD150 high HSCs can alleviate aging phenotypes in old recipient mice. Thus, our study demonstrates the presence of "younger" HSCs in old mice, and that aging-associated functional decline can be mitigated by reducing dysfunctional HSCs., Competing Interests: Competing interests: The authors declare the following competing financial interests: Y.Z. and Y.W. are the inventors of a patent related to the work presented in this manuscript. The patent number is 63472759 and the title is: “Compositions and Methods for Assessing, Treating, or Reducing Aging-Related Functional Decline”, which was filed on June 13, 2023., (© 2024. The Author(s).)

Background: The advent of autologous gene modified cell therapies to treat monogenic disorders has been a major step forward for the field of hematopoietic stem cell transplantation (HCT) and cellular therapies. The need for disease-specific conditioning to enable these products to provide a potential cure has required extrapolation from experience in myeloablative and non-myeloablative HCT for these disorders., Methods: In this manuscript, we review the current datasets and clinical experience using different conditioning regimens for autologous gene therapies in hemoglobinopathies, metabolic and lysosomal disorders, inborn errors of immunity (IEI) and bone marrow failure (BMF) syndromes., Results: The disease specific and unique conditioning requirements of each disorder are considered in order to achieve maximal benefit while minimizing associated toxicities., Conclusions: Standardized recommendations based on these data are made for each set of disorders to harmonize treatment. Future directions and the possibility of non-genotoxic conditioning regimens for autologous gene therapies are also discussed. Ethical Statement: The authors followed all relevant ethical considerations in writing this manuscript., Competing Interests: Declaration of Competing Interest Joseph Oved received consulting fees and/or honoraria from Emendo Bio, Turn.Bio, Grifols, Ensoma, Sobi and Amgen. He receives research support from Sobi and is the site PI for the Jasper SCID trial (NCT02963064). Akshay Sharma has received consultant fee from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, BioLineRx, Sangamo Therapeutics and Editas Medicine. He is a medical monitor for RCI BMT CSIDE clinical trial for which receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. Dr Sharma is the St. Jude Children's Research Hospital site principal investigator of clinical trials for genome editing of sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907) and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trial, which includes salary support paid to Dr Sharma's institution. Dr Sharma has no direct financial interest in these therapies. Amy E DeZern participated in advisory boards and/or had a consultancy with and received honoraria from Celgene/BMS, Agios, Novartis, Appellis and Gilead. AED served on clinical trial committees or DSMB for Novartis, Abbvie, Kura, Geron, Servier, Keros, Shuattuck labs and Celgene/BMS. SEP receives support for the conduct of clinical trials through Boston Children's Hospital from AlloVir, Atara, and Jasper Therapeutics. Honoraria from Pierre Fabre, and Regeneron. Consulting for Century, CellEvolve and VOR. Carmem Bonfim consults for Zodiac, Amgen and Novartis. Duncan Purtill receives honoraris (to Fiona Stanley Hospital and not the author) from Novartis, Gilead, BMS-Celgene and Jazz Pharmaceuticals. Sandeep Soni is employed by CRSPR Therapeutics AG. Jaap Jan Boelens consults for Advanced Clinical, Sanofi, Sobi, Immusoft, SmartImmune and Merck and receives research funding from Sanofi. Allistair Abraham served on the safety monitoring committee for Sangamo Therapeutics and has no financial interest in the development of gene therapies. Remaining authors have no conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. All rights reserved.)

The successful generation of long-term engrafting hematopoietic stem cells (HSCs) from human-induced pluripotent stem cells (hiPSCs) has long been sought to revolutionize treatments for hematological disorders, eliminating reliance on donors and avoiding immune rejection, and thus has been seen as a major milestone in regenerative medicine. Previous studies, guided by developmental hematopoiesis, made progress in creating blood cells from hiPSCs, but challenges persisted in producing hematopoietic cells with functional properties of genuine HSCs capable of long-term engraftment. In their recent study, Ng and colleagues described an optimized differentiation protocol that manipulates key signaling pathways, including TGF-β, WNT, BMP, and retinoic acid in a stage-specific manner to generate HSCs with multilineage capacity. This strategy yielded hematopoietic cells capable of engrafting long term with high levels of human chimerism in recipient mice. This research provides a blueprint for future studies aiming for personalized HSC-based therapies for various blood disorders.

Introduction: Autologous hematopoietic stem cell transplantation (ASCT) has gained extensive application in the treatment of lymphoma and multiple myeloma (MM). Plenty of studies demonstrate that peripheral blood indicators could be considered potential predictive biomarkers for hematopoietic stem cells (HSCs) collection efficiency, including white blood cell count (WBC), monocyte count (Mono), platelet count (PLT), hematocrit, and hemoglobin levels. Currently, clinically practical predictive models based on these peripheral detection indicators to quickly, conveniently, and accurately predict collection efficiency are lacking., Methods: In total, 139 patients with MM and lymphoma undergoing mobilization and collection of ASCT were retrospectively studied. The study endpoint was successful collection of autologous HSCs. We analyzed the effects of clinical characteristics and peripheral blood markers on collection success, and screened variables to establish a prediction model. We determined the optimal cutoff value of peripheral blood markers for predicting successful stem cell collection and the clinical value of a multi-marker prediction approach. We also established a prediction model for collection efficacy., Results: Univariate and multivariate logistic regression analyses showed that the mobilization regimen, Mono, PLT, mononuclear cell count (MNC), and peripheral blood CD34 + cell count (PB CD34 + counts) were significant predictors of successful collection of peripheral blood stem cells (PBSC). Two predictive models were constructed based on the results of multivariate logistic analyses. Model 1 included the mobilization regimen, Mono, PLT, and MNC, whereas Model 2 included the mobilization regimen, Mono, PLT, MNC, and PB CD34 + counts. Receiver operating characteristic (ROC) curve analysis showed that the PB CD34 + counts, Model 1, and Model 2 could predict successful HSCs collection, with cutoff values of 26.92 × 106/L, 0.548, and 0.355, respectively. Model 1 could predict successful HSCs collection with a sensitivity of 84.62%, specificity of 75.73%, and area under the curve (AUC) of 0.863. Model 2 could predict successful HSCs collection with a sensitivity of 83.52%, specificity of 94.17%, and AUC of 0.946; thus, it was superior to the PB CD34 + counts alone., Conclusion: Our findings suggest that the combination of the mobilization regimen, Mono, PLT, MNC, and PB CD34 + counts before collection has predictive value for the efficacy of autologous HSCs collection in patients with MM and lymphoma. Using models based on these predictive markers may help to avoid over-collection and improve patient outcomes., (© 2024 The Author(s). International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

The ability to perform hematopoietic cell transplant across major histocompatibility complex barriers can dramatically increase the availability of donors and allow more patients across the world to pursue curative transplant procedures for underlying hematologic disorders. Early attempts at haploidentical transplantation using broadly reactive T-cell depletion approaches were compromised by graft rejection, graft-versus-host disease and prolonged immune deficiency. The evolution of haploidentical transplantation focused on expanding transplanted hematopoietic progenitors as well as using less broadly reactive T-cell depletion. Significant outcome improvements were identified with technology advances allowing selective depletion of donor allospecific T cells, initially ex-vivo with evolution to its current in-vivo approach with the infusion of the highly immunosuppressive chemotherapy agent, cyclophosphamide after transplantation procedure. Current approaches are facile and portable, allowing expansion of allogeneic hematopoietic cell transplantation for patients across the world, including previously underserved populations.

Haematopoietic stem and progenitor cell (HSPC) autologous gene therapies are promising treatment for a variety of blood disorders. Investigation of the long-term HSPC clonal dynamics and other measures of safety and durability following lentiviral-mediated gene therapies in predictive models are crucial for assessing risks and benefits in order to inform decisions regarding wider utilization. We established an autologous lentivirally barcoded HSPC transplantation model in rhesus macaque (RM), a model offering insights into haematopoiesis and gene therapies with direct relevance to human. Healthy young adult RMs underwent total body irradiation, followed by transplantation of autologous HSPCs transduced with a lentiviral vector containing a diverse genetic barcode library, uniquely labelling individual HSPCs and their progeny. With up to 131 months of follow-up, we now report quantitative clonal dynamics, characterizing the number, diversity, stability and lineage bias of hundreds of thousands of HSPC clones tracked in five RMs. We documented long-term stable and multi-lineage output from a highly polyclonal pool of HSPCs. Clonal succession after stable haematopoietic reconstitution was minimal. There was no evidence for accelerated acquisition of acquired somatic mutations following autologous lentivirally transduced HSPC transplantation. Our results provide relevant insights into long-term HSPC behaviours in vivo following transplantation and gene therapies., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells have become the preferred source of hematopoietic stem cells. We compared the effectiveness of G-CSF and pegylated G-CSF (peg-G-CSF) for hematopoietic stem cell mobilization in haploidentical hematopoietic stem cell transplantation (haplo-HSCT) donors, and evaluated the transplant outcomes. We conducted a matched retrospective cohort study. Donors mobilized with peg-G-CSF (n = 70) and G-CSF (n = 70). 140 consecutive patients diagnosed with acute leukemia who underwent haplo-HSCT were included in this study. The findings revealed that the peg-G-CSF cohort exhibited significantly elevated myeloid-derived suppressor cells (MDSCs) levels in their grafts when compared to the G-CSF cohort (P < 0.001). The 100-day cumulative incidence (CI) of grade III-IV acute graft-versus-host disease (GVHD) and 1-year CI of moderate-to-severe chronic GVHD were 4.3% vs 14.3 % (P = 0.047) and 11.2% vs 27.4 % (P = 0.023), in the peg-G-CSF group and G-CSF group. Patients reveiving mobilized stem cell with peg-G-CSF had a significantly greater likelihood of 1-year GVHD-free relapse-free survival (GRFS) compared to patients reveiving mobilized stem cell with G-CSF (74.9% vs 37.9 %, P < 0.001). The higher graft MDSCs proportion was associated with lower grade II-IV aGVHD, cGVHD (P < 0.05) and higher GRFS in the univariate analysis (P < 0.05). Multivariate analysis showed that MDSCs proportion higher than 11.36 % (HR, 0.305; 95 % CI, 0.154-0.606; P = 0.001) and peg-G-CSF for stem cell mobilization (HR, 0.466; 95 % CI, 0.251-0.865; P = 0.016) were independent prognostic factors of GRFS. The superior survival rates observed in recipients of peg-G-CSF-mobilized cells are likely due to reduced acute GVHD, potentially mediated by the increased MDSCs within the grafts., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Haematopoietic stem cell (HSC) gene therapy (GT) may provide lifelong reconstitution of the haematopoietic system with gene-corrected cells 1 . However, the effects of underlying genetic diseases, replication stress and ageing on haematopoietic reconstitution and lineage specification remain unclear. In this study, we analysed haematopoietic reconstitution in 53 patients treated with lentiviral-HSC-GT for diverse conditions such as metachromatic leukodystrophy 2,3 (MLD), Wiskott-Aldrich syndrome 4,5 (WAS) and β-thalassaemia 6 (β-Thal) over a follow-up period of up to 8 years, using vector integration sites as markers of clonal identity. We found that long-term haematopoietic reconstitution was supported by 770 to 35,000 active HSCs. Whereas 50% of transplanted clones demonstrated multi-lineage potential across all conditions, the remaining clones showed a disease-specific preferential lineage output and long-term commitment: myeloid for MLD, lymphoid for WAS and erythroid for β-Thal, particularly in adult patients. Our results indicate that HSC clonogenic activity, lineage output, long-term lineage commitment and rates of somatic mutations are influenced by the underlying disease, patient age at the time of therapy, the extent of genetic defect correction and the haematopoietic stress imposed by the inherited disease. This suggests that HSCs adapt to the pathological condition during haematopoietic reconstitution., Competing Interests: Competing interests: The San Raffaele Telethon Institute for GT (SR-Tiget) is a joint venture between the Telethon Foundation and Ospedale San Raffaele. Lentiviral vector-based gene therapy for metachromatic leukodystrophy (MLD), developed at SR-Tiget, was licensed to Orchard Therapeutics in 2018. Lentiviral vector-based gene therapy for Wiskott–Aldrich (WAS) syndrome was developed by Fondazione Telethon. Lentiviral vector-based gene therapy for β-thalassemia was developed at SR-Tiget. Gene therapy for MLD is approved in the EU (Libmeldy) and in the US (Lenmeldy). A.A. was the principal investigator of the pilot and pivotal SR-Tiget clinical trial of GT for MLD, WAS and β-Thal. The other authors declare no competing interests., (© 2024. The Author(s).)

Background: Hematopoietic stem cell transplantation (HSCT) is one of the most effective ways to treat hematological malignant diseases, but the traditional culture of hematopoietic stem cells (HSCs) in vitro will soon lose their ability to self-renewal or differentiate into multilineage blood cells., Methods: To determine whether Forkhead boxO1 (FoxO1) is implicated in the development of HSCs, lentiviral vectors expressing knockdown (KD) or overexpression (OE) of FoxO1 were utilized in fetal liver-derived hematopoietic stem and progenitor cells (FL-HSPCs). The impacts on the proliferation and hematopoietic differentiation of FL-HSPCs were subsequently evaluated via flow cytometry (FCM). Furthermore, the effect of FoxO1-OE on the self-renewal of cord blood-derived hematopoietic stem and progenitor cells (CB-HSPCs) was investigated. Additionally, the transplantation ability of hematopoietic stem cells derived from these CB-HSPCs in mice after secondary transplantation was also assessed by FCM., Result: After knocking down FoxO1 in FL-HSPCs, the apoptosis rate was significantly increased, and the expression of hCD45 was significantly decreased. Conversely, overexpression of FoxO1 reversed this phenomenon, effectively promoting the expansion and differentiation of FL-HSPCs in vitro. Similarly, it was found that FoxO1-OE could effectively enhance the expansion of CB-HSPCs. Furthermore, upon transplantation of CB-HSPCs overexpressing FoxO1 into NSG mice, multilineage human hematopoietic reconstruction was promoted. Notably, the results of secondary transplantation revealed that only the FoxO1-OE group exhibited multilineage reconstitution., Conclusion: In conclusion, our study confirmed that FoxO1-OE could enhance the self-renewal and engraftment of CB-HSPCs., Competing Interests: Declarations. Ethics approval and consent to participate: All animal procedures were carried out in compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and ARRIVE guidelines. The study was approved Medical Ethics Committee of The First Affiliated Hospital of Ningbo University (2024-R054-01). Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Abstract: Hematopoietic stem cell (HSC) transplantation with lentiviral vector (LVV)-transduced autologous cells has proven an effective therapeutic strategy for sickle cell disease (SCD). However, ex vivo culture or proliferative stress associated with in vivo reconstitution may amplify any underlying genetic risk of leukemia. We aimed to minimize culture-induced stress and reduce genomic damage during ex vivo culture and enhance stem cell fitness and reconstitution of SCD CD34+ cells transduced with BCL11A shmiR-encoding LVV. UM171, a pyrimidoindole derivative, can expand normal HSCs during in vitro culture and has been shown to be safe and effective using umbilical cord blood. We examined the effect of UM171 during ex vivo LVV transduction of SCD HSCs. Culture of SCD CD34+ HSCs with UM171 during transduction reduced DNA damage and reactive oxygen species, decreased apoptosis, and was associated with increased numbers of immunophenotypically defined long-term HSCs. UM171 increased the engraftment of LVV-transduced human HSCs in immunodeficient mice and barcode tracing revealed increased clonal diversity of engrafting cells. In competitive transplantation assays, analysis of bone marrow showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions. In summary, exposure of SCD peripheral blood CD34+ cells to UM171 during LVV transduction enhances stem cell fitness. These findings suggest manufacturing of genetically modified HSCs in the presence of UM171 may improve efficacy, safety, and sustainability of gene therapy using ex vivo approaches. BCL11A shmiR-encoding LVV is in clinical trials to treat SCD (NCT03282656), UM171 is in clinical trials to culture umbilical cord blood (NCT02668315)., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 -/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence, we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice, and in lineage specific, regulated and robust transgene expression. This approach, which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells, may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases., Competing Interests: Competing interests: A.B., A.M., and P.G. are co-authors in the provisional patent application PCT/US23/22029. A.B. and A.M. are co-authors in the patent PCT/US20/45106. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Purpose of Review: Stem cell donor registries play an important role in providing stem cell products from unrelated donors to patients with severe blood diseases. In this review, important aspects of donor registry work, current challenges and possible future developments are discussed., Recent Findings: The current growth in global unrelated stem cell donations is in line with the long-term trend, indicating that donor registries have overcome the COVID-19 pandemic. A key challenge for donor registries is the recruitment of donors from disadvantaged populations to create greater equity in access to unrelated stem cell transplantation. In addition, recruiting young donors and increasing the availability of donors who are already registered are important goals. In recent years, numerous studies have looked at the context of these themes and the development of possible solutions., Summary: The international community of donor registries, together with the World Marrow Donor Association, has helped many patients in need of a stem cell transplant over the past decades and is, therefore, a bright example of international collaboration for a good cause. It is currently addressing a number of challenges to effectively help as many patients as possible from various populations also in the future., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent β-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions., Competing Interests: Declaration of competing interest Ashish Gupta is a site principal investigator at University of Minnesota for clinical trials in genome editing for sickle cell disease sponsored by Bluebird Bio (NCT04293185) and Beam Therapeutics (NCT05456880). He is also the site principal investigator at University of Minnesota for clinical trials in genome editing for Hurler syndrome (NCT06149403). He has received consultancy fees from Vertex Pharmaceuticals and is on speaker bureau for Emerging Therapies Solutions. He also has research funding from Jazz Pharmaceuticals for an investigator initiated trial. Akshay Sharma has received consultant fees from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, Sangamo Therapeutics and Editas Medicine. He is a medical monitor for an RCI BMT clinical trial for which he receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. Akshay Sharma is also the St. Jude Children's Research Hospital site principal investigator for clinical trials of genome editing for sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907) and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trials, which includes salary support paid to the investigator's institution. Akshay Sharma has no direct financial interest in these therapies. Jaap Jan Boelens has received consultant fees from Sanofi, Sobi, Merck, Immusoft and Smart Immune, is the MSKCC site PI for Novartis Pharmaceuticals (NCT04443907) and received research funding from Sanofi (for investigator-initiated research). Jaap Jan Boelens is also the chair of an independent data safety monitoring board (for bluebird bio gene therapy trials), for which he receives compensation (CRO; Advanced Clinical). Susan Prockop has received support for the conduct of clinical trials from AlloVir, Atara, and Jasper through Boston Children's Hospital. She has received an honorarium from Pierre Fabre and Regeneron and consults for CellEvolve, Smart Immune and Century. She has IP related to the implementation of third-party viral-specific T cells, with all rights assigned to Memorial Sloan Kettering Cancer Center. Chrystal U. Louis has equity in and is an employee of TScan Therapeutics. The other authors have nothing to disclose., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those from a donor 1,2 . Here, to quantify dynamics of long-term stem cell engraftment, we sequenced genomes from 2,824 single-cell-derived haematopoietic colonies of ten donor-recipient pairs taken 9-31 years after HLA-matched sibling HCT 3 . With younger donors (18-47 years at transplant), 5,000-30,000 stem cells had engrafted and were still contributing to haematopoiesis at the time of sampling; estimates were tenfold lower with older donors (50-66 years). Engrafted cells made multilineage contributions to myeloid, B lymphoid and T lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to around 10-15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. A transplant-related population bottleneck could not explain these differences; instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In pruning selection, cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant-their selective advantage derived from preferential mobilization, collection, survival ex vivo or initial homing. In growth selection, cell divisions underpinning clonal expansion occurred in the recipient's marrow after engraftment, most pronounced in clones with multiple driver mutations. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors., Competing Interests: Competing interests: P.J.C. is a co-founder, stock holder and consultant for Quotient Therapeutics., (© 2024. The Author(s).)

Humanized mice, defined as mice with human immune systems, have become an emerging model to study human hematopoiesis, infectious disease, and cancer. Here, we describe the techniques to generate humanized NSGF6 mice using adult human CD34 + hematopoietic stem and progenitor cells (HSPCs). We describe steps for constructing and monitoring the engraftment of humanized mice. We then detail procedures for tissue processing and immunophenotyping by flow cytometry to evaluate the multilineage hematopoietic differentiation. For complete details on the use and execution of this protocol, please refer to Yu et al. 1 ., Competing Interests: Declaration of interests C.I.Y., R.M., J.B., and K.P. filed a patent on novel humanized mouse models via genetic editing of NSG mouse. K.P. is a stockholder in Cue Biopharma and Guardian Bio, scientific advisor to Cue Biopharma and Guardian Bio, and co-founder of Guardian Bio. K.P. declares unrelated funding support from Guardian Bio (current) and Merck (past). J.B. is associated with Immunai., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Umbilical cord blood (UCB) is a rich source of hematopoietic stem and progenitor cells that are biologically superior to their adult counterparts. UCB cells can be stored for several years without compromising their numbers or function. Today, public and private UCB banks have been established in several countries around the world. After 35 years since the first UCB transplant (UCBT), more than 50,000 UCBTs have been performed worldwide. In pediatric patients, UCBT is comparable to or superior to bone marrow transplantation. In adult patients, UCB can be an alternative source of hematopoietic cells when an HLA-matched unrelated adult donor is not available and when a transplant is urgently needed. Delayed engraftment (due to reduced absolute numbers of hematopoietic cells) and higher costs have led many medical institutions not to consider UCB as a first-line cell source for hematopoietic transplants. As a result, the use of UCB as a source of hematopoietic stem and progenitor cells for transplantation has declined over the past decade. Several approaches are being investigated to make UCBTs more efficient, including improving the homing capabilities of primitive UCB cells and increasing the number of hematopoietic cells to be infused. Several of these approaches have already been applied in the clinic with promising results. UCB also contains immune effector cells, including monocytes and various lymphocyte subsets, which, together with stem and progenitor cells, are excellent candidates for the development of cellular therapies for hematological and non-hematological diseases., (Copyright © 2024 Instituto Mexicano del Seguro Social (IMSS). Published by Elsevier Inc. All rights reserved.)

Abstract: Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the Beige and Chediak-Higashi domain-containing protein (BDCP), Neurobeachin (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LPS Responsive Beige-Like Anchor Protein (LRBA), Neurobeachin Like 1 (NBEAL1) and Lysosomal Trafficking Regulator (LYST), are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block after transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In summary, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model in which BDCP-regulated vesicle trafficking is required for efficient HSCT., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

It has been proposed that adult hematopoiesis is sustained by multipotent progenitors (MPPs) specified during embryogenesis. Adult-like hematopoietic stem cell (HSC) and MPP immunophenotypes are present in the fetus, but knowledge of their functional capacity is incomplete. We found that fetal MPP populations were functionally similar to adult cells, albeit with some differences in lymphoid output. Clonal assessment revealed that lineage biases arose from differences in patterns of single-/bi-lineage differentiation. Long-term (LT)- and short-term (ST)-HSC populations were distinguished from MPPs according to capacity for clonal multilineage differentiation. We discovered that a large cohort of long-term repopulating units (LT-RUs) resides within the ST-HSC population; a significant portion of these were labeled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will significantly underestimate the size and diversity of the LT-RU pool and (2) LT-RUs in the ST-HSC population have the attributes required to persist into adulthood., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

There is lack of guidance for immune monitoring and infection prevention after administration of ex vivo genetically modified hematopoietic stem cell therapies (GMHSCT). We reviewed current infection prevention practices as reported by providers experienced with GMHSCTs across North America and Europe, and assessed potential immunologic compromise associated with the therapeutic process of GMHSCTs described to date. Based on these assessments, and with consensus from members of the International Society for Cell & Gene Therapy (ISCT) Stem Cell Engineering Committee, we propose risk-adapted recommendations for immune monitoring, infection surveillance and prophylaxis, and revaccination after receipt of GMHSCTs. Disease-specific and GMHSCT-specific considerations should guide decision making for each therapy., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34 + HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45 + cells in the peripheral blood, even when only 5,000-10,000 CD34 + HSCs were transferred. Efficient engraftment of human CD45 + cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34 + CD38 - or CD34 + CD38 + cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45 + and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34 + cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility., (© 2024. The Author(s).)

The purpose of this review is to summarize the current understanding of the therapeutic effect of stem cell-based therapies, including hematopoietic stem cells, for the treatment of ischemic heart damage. Following PRISMA guidelines, we conducted electronic searches in MEDLINE, and EMBASE. We screened 592 studies, and included RCTs, observational studies, and cohort studies that examined the effect of hematopoietic stem cell therapy in adult patients with heart failure. Studies that involved pediatric patients, mesenchymal stem cell therapy, and non-heart failure (HF) studies were excluded from our review. Out of the 592 studies, 7 studies met our inclusion criteria. Overall, administration of hematopoietic stem cells (via intracoronary or myocardial infarct) led to positive cardiac outcomes such as improvements in pathological left-ventricular remodeling, perfusion following acute myocardial infarction, and NYHA symptom class. Additionally, combined death, rehospitalization for heart failure, and infarction were significantly lower in patients treated with bone marrow-derived hematopoietic stem cells. Our review demonstrates that hematopoietic stem cell administration can lead to positive cardiac outcomes for HF patients. Future studies should aim to increase female representation and non-ischemic HF patients.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier Inc.)

Background/aim: The COVID-19 pandemic has had a significant impact on the current management of allotransplanted patients in whom fresh hematopoietic stem cells (HSCs) were replaced by cryopreserved ones. The aim of the study was to determine the efficacy and safety of cryopreserved HSCs when compared with the fresh ones., Patients and Methods: A retrospective analysis of 254 allogeneic stem cell transplantations (HSCT) procedures performed between 2020-2021 included the following donors: matched related (MRD; n=68), matched unrelated (MUD; n=148) and haploidentical (HID; n=38). 50% of patients (non-cryo group) received fresh grafts, whereas the remaining patients (cryo group) were transplanted with cryopreserved cells., Results: No differences in terms of median days to neutrophil [MRD/MUD/HID cryo- and non-cryo groups: 17 vs. 16 (p=0.27), 19 vs. 18 (p=0.83), 22 vs. 22 (p=0.83) days, respectively] and platelet [MRD/MUD/HID cryo- and non-cryo groups: 14 vs. 14 (p=0.25), 17 vs. 17 (p=0.33), 21 vs. 19 (p=0.36) days, respectively] engraftments were demonstrated. Among MUD graft recipients, platelet engraftment rates were 81% in the cryo- and 96% in the non-cryo group (p=0.01). OS rates were comparable at 1 year after HSCT between MRD/MUD/HID cryo- and non-cryo groups: 53% vs. 60% (p=0.54), 60% vs. 66% (p=0.5), 50% vs. 41% (p=0.56), respectively., Conclusion: During the COVID-19 pandemic, cryopreserved HSCs did not have a negative impact on median engraftment time and OS when compared to fresh HSCs. In the MUD group, platelet engraftment rate was lower in cryopreserved HSC recipients., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background Aims: Cryopreservation of hematopoietic stem cells (HSCs) is crucial for autologous transplantation, cord blood banking and other special circumstances. Dimethyl sulfoxide (DMSO) is used most commonly for cryopreserving HSC products but can cause infusional toxicities and affect cell viability and engraftment after transplant. A systematic review of controlled studies using lower concentrations of DMSO to cryopreserve HSC products in clinical transplant studies is needed to determine the effect of reducing DMSO concentrations on post-thaw cell viability, initial engraftment and adverse effects on patient health., Methods: All studies identified in our systematic search (to July 11, 2023) examining the use of cryopreserved peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (AHCT) were included. Meta-analysis was performed to determine how varying the concentration of DMSO during cryopreservation effects post-thaw cell viability, initial engraftment and adverse effects on patient health., Results: A total of 1547 studies were identified in our systematic search, with seven published articles meeting eligibility for inclusion in meta-analysis. All patients underwent AHCT using (PBSCs) to treat hematologic malignancies. The viability of CD34+ cells post thaw was greater when cryopreserved with 5% DMSO compared with 10% DMSO, with lower rates of adverse side effects in patients. DMSO concentration had minimal impact on rates of initial engraftment. Significant heterogeneity in outcome reporting was observed and the potential for bias was identified in all studies., Conclusions: Reducing the concentration of DMSO from 10% to 5% during cryopreservation of autologous PBSCs may improve cell viability and reduce DMSO-associated adverse effects in patients undergoing AHCT. Data from more studies with similar patients and standard outcome reporting are needed to increase confidence in our initial observations., Protocol Registration: PROSPERO; registration number CRD42023476809 registered November 8, 2023., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Abstract: Autophagy is an intracellular survival process that has established roles in the long-term survival and function of hematopoietic stem cells (HSC). We investigated the contribution of autophagy to HSC fitness during allogeneic transplantation and graft-versus-host disease (GVHD). We demonstrate in vitro that both tumor necrosis factor and IL-1β, major components of GVHD cytokine storm, synergistically promote autophagy in both HSC and their more mature hematopoietic progenitor cells (HPC). In vivo we demonstrate that autophagy is increased in donor HSC and HPC during GVHD. Competitive transplant experiments demonstrated that autophagy-deficient cells display reduced capacity to reconstitute the hematopoietic system compared to wild-type counterparts. In a major histocompatibility complex-mismatched model of GVHD and associated cytokine dysregulation, we demonstrate that autophagy-deficient HSC and progenitors fail to establish durable hematopoiesis, leading to primary graft failure and universal transplant related mortality. Using several different models, we confirm that autophagy activity is increased in early progenitor and HSC populations in the presence of T-cell-derived inflammatory cytokines and that these HSC populations require autophagy to survive. Thus, autophagy serves as a key survival mechanism in HSC and progenitor populations after allogeneic stem cell transplant and may represent a therapeutic target to prevent graft failure during GVHD., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Objective: To investigate the efficiency and optimal time of stem cell apheresis mobilized by pegylated recombinant human granulocyte colony stimulating factor (PEG-rhG-CSF) in autologous stem cell transplantation (ASCT) for hematological malignancies without monitoring pre-collection CD34 + cells., Methods: Forty-six patients underwent stem cell mobilization were retrospectively analyzed between August 2017 and January 2022 at the First Affiliated Hospital of Fujian Medical University. 27 patients using high dose chemotherapy combined with PEG-rhG-CSF mobilization were enrolled in the PEG-rhG-CSF group, and other 19 patients mobilized with recombinant human granulocyte colony stimulating factor (G-CSF) were enrolled in G-CSF group. The mobilization and collection effects of the patients in two groups were compared., Results: A total of 46 patients underwent 86 apheresis procedures, the median amount of mononuclear cell (MNC) in the PEG-rhG-CSF group and G-CSF group was 6.54（3.85-12.61）×10 8 /kg and 6.15（1.13-11.58）×10 8 /kg, respectively ( P >0.05), the total CD34 + cells of the grafts were 11.44(1.33-65.02)×10 6 /kg and 4.95(0.30-24.02)×10 6 /kg ( P < 0.05), with harvest timing of 14(10-20) days and 14(4-22) days, respectively ( P >0.05). In the PEG-rhG-CSF group, there was a significant difference between the number of CD34 + cells collected when white blood cells (WBC) ≥10×10 9 /L and WBC<10×10 9 /L, 19.04(2.85-65.02)×10 6 /kg and 6.22(0.81-34.86)×10 6 /kg, respectively ( P < 0.05)., Conclusion: Stem cells mobilization with PEG-rhG-CSF was highly efficient with a median mobilization time of 14 days. In the absence of peripheral blood CD34 monitoring, peripheral blood WBC≥10×10 9 /L can be considered as a threshold for a single stem cell apheresis to collect sufficient stem cells.

Hematopoietic stem cell (HSC)-targeted gene therapy is curative for various genetic blood diseases, and its efficacy has been demonstrated in recent clinical trials. HSCs have self-renewal and hematopoietic multipotency; therefore, repairing pathological mutations or defects in HSCs allows for a lifelong cure with a single treatment. Autologous HSC gene therapy has been developed by lentiviral gene addition or gene editing, and is an option for most patients because it does not require a compatible donor. Current HSC gene therapy is based on ex vivo methods, in which patient HSCs are harvested, genetically modified ex vivo, and autologously transplanted into patients. However, the complexity of this process and the high cost of treatment are hindering the spread of gene therapy. Therefore, in vivo HSC gene therapy is being developed to deliver gene therapy tools directly into bone marrow HSCs by administration without ex vivo culture.

Historically, defining haematopoietic subsets, including self-renewal, differentiation and lineage restriction, has been elucidated by transplanting a small number of candidate cells with many supporting bone marrow (BM) cells. While this approach has been invaluable in characterising numerous distinct subsets in haematopoiesis, this approach is arguably flawed. The haematopoietic stem cell (HSC) has been proposed as the critical haematopoietic subset necessary for transplantation. However, due to the presence of supporting cells, the HSC has never demonstrated sufficiency. Utilising the homeobox B5 (Hoxb5)-reporter system, we found that neither long-term (LT) HSCs nor short-term (ST) HSCs alone were sufficient for long-term haematopoietic reconstitution. Critically, reconstitution can be rescued by transplanting combined LT- and ST-HSCs, without supporting cells; a fraction we term the 'Minimum Subset for Transplantation' (MST). The MST accounts for only 0·005% of nucleated cells within mouse BM, and this MST can be cultured, expanded and genetically modified while preserving its rapid haematopoietic engraftment potential. These results support the consideration of an MST approach for clinical translation, especially for gene therapy approaches that require HSC compartment modification., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-Prkdc scid IL2rg tm1Wjl /SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments., (©2021 Society for Leukocyte Biology.)

Recent evidence indicates that extracellular adenosine triphosphate (eATP), as a major mediator of purinergic signaling, plays an important role in regulating the mobilization and homing of hematopoietic stem progenitor cells (HSPCs). In our previous work we demonstrated that eATP activates the P2X7 ion channel receptor in HSPCs and that its deficiency impairs stem cell trafficking. To learn more about the role of the P2X purinergic receptor family in hematopoiesis, we phenotyped murine and human HSPCs with respect to the seven P2X receptors and observed that, these cells also highly express P2X4 receptors, which shows ~50% sequence similarity to P2X7 subtypes, but that P2X4 cells are more sensitive to eATP and signal much more rapidly. Using the selective P2X4 receptor antagonist PSB12054 as well as P2X4-KO mice, we found that the P2X4 receptor, similar to P2X7 receptor, promotes trafficking of HSPCs in that its deficiency leads to impaired chemotaxis of HSPCs in response to a stromal-derived factor 1 (SDF-1) gradient, less effective pharmacological mobilization, and defective homing and engraftment of HSPCs after transplantation into myeloablated hosts. This correlated with a decrease in SDF-1 expression in the BM microenvironment. Overall, our results confirm the proposed cooperative dependence of both receptors in response to eATP signaling. In G-CSF-induced mobilization, a lack of one receptor is not compensated by the presence of the other one, which supports their mutual dependence in regulating HSPC trafficking., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

During 2020, the concurrent novel COVID-19 pandemic lead to widespread cryopreservation of allogeneic hematopoietic cell transplant grafts based on National Marrow Donor Program and European Society of Blood and Marrow Transplantation recommendations, in order to secure grafts before the start of conditioning chemotherapy. We sought to examine the impact of this change in practice on patient outcomes. We analyzed the outcomes of 483 patients who received hematopoietic stem cell transplantation (HSCT) between August 2017 and August 2020, at Princess Margaret Cancer Centre, Canada, in the retrospective study, comparing the outcomes between those who received cryopreserved or fresh peripheral blood stem cell grafts. Overall compared with those who received fresh grafts (n = 348), patients who received cryopreserved grafts (n = 135) had reduced survival and GRFS, reduced incidence of chronic graft-versus-host disease (GvHD), delay in neutrophil engraftment, and higher graft failure (GF), with no significant difference in relapse incidence or acute GvHD. However, recipients of cryopreserved matched-related donor HSCT showed significantly worse OS, NRM, GRFS compared with fresh grafts. Multivariable analysis of the entire cohort showed significant impact of cryopreservation on OS, relapse, cGvHD, GF, and GRFS. We conclude that cryopreservation was associated with inferior outcomes post-HSCT, possibly due to the combination of ATG and post-transplant cyclophosphamide impacting differential tolerance to cryopreservation on components of the stem cell graft; further studies are warranted to elucidate mechanisms for this observation., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hematopoietic stem cell (HSCs) transplantation is the primary therapeutic modality used to treat hematopoietic disorders. It centers on the capability of a small quantity of HSCs to repopulate whole blood lineages. Along with limited availability of suitable donors, the need for sufficient number of donor HSCs is still challenging in clinical relevance. This has been addressed by ex vivo HSC expansion albeit with partial success, and thus development of an alternative strategy that could improve HSC expansion is required. To that end, we aimed to build HematoMiR, an oligo-based technology that broadly targets HSC quiescence factors. Here, we show that HematoMiRs and their combinations targeting over 50 factors involved in HSC quiescence could induce robust ex vivo murine and human HSC expansion. In particular, HematoMiR-5 treatment enhanced cell cycle through down-regulation of negative cell cycle regulators in HSCs. HematoMiR-5 treated HSPCs had reduced DNA damage during the course of ex vivo expansion. Moreover, HematoMiR-5 treatment led to sustained HSC self-renewal ability and a low apoptosis rate. In addition, HematoMiR-5 expanded HSCs demonstrated successful engraftment and repopulation capacity in the recipient animals. Furthermore, combinatorial treatments of HematoMiR-2 and 5 allowed vigorous ex vivo HSC expansion. These findings demonstrate that novel and synthetic HematoMiR technology is feasible for HSC ex vivo expansion through the sequence-dependent modulation of numerous HSC quiescence modulators., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns -/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk -/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk -/- -HSPCs into Ctns -/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects., Competing Interests: Declaration of Competing Interest Stephanie Cherqui is co-inventor on a patent entitled “Methods of treating lysosomal disorders” (#20378–101,530), and is a cofounder, shareholder and a member of both the Scientific Board and board of directors of Papillon Therapeutics Inc. Stephanie Cherqui serves as a consultant for AVROBIO and receives compensation for these services. Stephanie Cherqui also serves as a member of the Scientific Review Board and Board of Trustees of the Cystinosis Research Foundation. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) have been used for therapeutic purposes for decades in the form of autologous and allogeneic transplantation and are currently emerging as an attractive target for gene therapy. A low stem cell dose is a major barrier to the application of HSC therapy in several situations, primarily umbilical cord blood transplantation and gene modification. Strategies that promote ex vivo expansion of the numbers of functional HSCs could overcome this barrier, hence have been the subject of intense and prolonged research. Several ex vivo expansion strategies have advanced to evaluation clinical trials, which are showing favorable outcomes along with convincing safety signals. Preclinical studies have recently confirmed beneficial incorporation of ex vivo expansion into HSC gene modification protocols. Collectively, ex vivo HSC expansion holds promise for significantly broadening the availability of cord blood units for transplantation, and for optimizing gene therapy protocols to enable their clinical application., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45 dim Lin - CD34 + cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution., (© 2021. The Author(s).)

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for hematopoietic malignancies. The therapeutic effect relies on donor T cells and NK cells to recognize and eliminate malignant cells, known as the graft-versus-leukemia (GVL) effect. However, off target immune pathology, known as graft-versus-host disease (GVHD) remains a major complication of alloSCT that limits the broad application of this therapy. The presentation of recipient-origin alloantigen to donor T cells is the primary process initiating GVHD and GVL. Therefore, the understanding of spatial and temporal characteristics of alloantigen presentation is pivotal to attempts to separate beneficial GVL effects from detrimental GVHD. In this review, we discuss mouse models and the tools therein, that permit the quantification of alloantigen presentation after alloSCT., Competing Interests: GRH has consulted for Generon Corporation, NapaJen Pharma, Neoleukin Therapeutics. iTeos Therapeutics and has received research funding from Roche, Compass Therapeutics, Syndax Pharmaceuticals, Applied Molecular Transport and iTeos Therapeutics., (Copyright © 2021 Koyama and Hill.)

Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation., (Copyright © 2021. Published by Elsevier Inc.)

Background: Collection of a large number of early hematopoietic progenitors is essential for allogeneic apheresis products intended for TCR-alpha/beta depletion., Materials and Methods: We added plerixafor 0.24 mg/kg body weight (bw) on day 4 of high-dose filgrastim mobilization 10 hours prior to apheresis in 16 (30.5%) pediatric allogeneic donors who failed to recover a sufficient number of CD34+ cells., Results: On day 4 of G-CSF, the median CD34+ cell count in peripheral blood was 6 per μL (range 4-9 per μL) in 6 poor mobilizers and 16 per μL (range 12-19 per μL) in insufficient mobilizers. In all donors, the threshold of 50 CD34+ cells/μL was achieved, and the median increase was 14.8-fold in poor mobilizers and 6.5-fold in insufficient mobilizers, whereas it was 3.45-fold increase in those mobilized with G-CSF alone., Discussion: In all donors, a predefined number of >10 × 10 6 CD34+ cells/kg of recipient bw before depletion was reached in the apheresis product. The use of plerixafor did not affect the purity of further TCR-alpha/beta depletion. Side effects were mild to moderate and consisted of nausea and vomiting., Conclusion: Thus, the safety and high efficacy of plerixafor was proven in healthy pediatric allogeneic hematopoietic cell donors., (© 2021 Wiley Periodicals LLC.)

Hematopoietic stem cells (HSCs) are typically characterized by transplantation into irradiated hosts in a highly perturbed microenvironment. Here, we show that selective and temporally controlled depletion of resident HSCs through genetic deletion of Gata2 constitutes efficient recipient conditioning for transplantation without irradiation. Strikingly, we achieved robust engraftment of donor HSCs even when delaying Gata2 deletion until 4 weeks after transplantation, allowing homing and early localization to occur in a completely non-perturbed environment. When HSCs from the congenic strains Ly5.1 and Ly5.2 were competitively transplanted, we found that the more proliferative state of Ly5.2 HSCs was associated with superior long-term engraftment when using conditioning by standard irradiation, while higher CXCR4 expression and a better homing ability of Ly5.1 HSCs strongly favored the outcome in our inducible HSC depletion model. Thus, the mode and timing of recipient conditioning challenges distinct functional features of transplanted HSCs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB 1 . We used a custom adenine base editor (ABE8e-NRCH) 2,3 to convert the SCD allele (HBB S ) into Makassar β-globin (HBB G ), a non-pathogenic variant 4,5 . Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB S to HBB G . Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB G was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB S base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse 6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB S -to-HBB G editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB S , generates benign HBB G , and minimizes the undesired consequences of double-strand DNA breaks.

Hereditary anemia has various manifestations, such as sickle cell disease (SCD), Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency (G6PDD), and thalassemia. The available management strategies for these disorders are still unsatisfactory and do not eliminate the main causes. As genetic aberrations are the main causes of all forms of hereditary anemia, the optimal approach involves repairing the defective gene, possibly through the transplantation of normal hematopoietic stem cells (HSCs) from a normal matching donor or through gene therapy approaches (either in vivo or ex vivo) to correct the patient's HSCs. To clearly illustrate the importance of cell and gene therapy in hereditary anemia, this paper provides a review of the genetic aberration, epidemiology, clinical features, current management, and cell and gene therapy endeavors related to SCD, thalassemia, Fanconi anemia, and G6PDD. Moreover, we expound the future research direction of HSC derivation from induced pluripotent stem cells (iPSCs), strategies to edit HSCs, gene therapy risk mitigation, and their clinical perspectives. In conclusion, gene-corrected hematopoietic stem cell transplantation has promising outcomes for SCD, Fanconi anemia, and thalassemia, and it may overcome the limitation of the source of allogenic bone marrow transplantation.

Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing (∼21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs., (Published by Elsevier Inc.)

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.

Aging affects the functionality of hematopoietic stem cells (HSCs), and therefore, aged individuals are not preferred as donors in HSC transplantation. Such elimination leads to the restriction of donor cohort. Several efforts are being done to rejuvenate aged HSCs. Here, we show that treatment of aged mice with curcumin rejuvenates their HSCs by restoring the expression of autophagy-inducing messenger RNAs in them, and improves their engraftment capacity. Importantly, we show that curcumin is effective in rejuvenation of HSCs when administered via both, intraperitoneal as well as oral routes. Aging also affects the immune system. While elderly individuals are not immuno-deficient, they do not respond optimally to immunizations, and hence, a strategy needs to be developed to make them immunologically responsive. Programmed cell death 1 (PD-1), one of the inhibitory coreceptors, plays an important role in the regulation of autoimmunity, infectious immunity, and cancer immunity. Its expression on T cells is indicative of their exhaustion. Here, we show that curcumin reduces the frequency of PD1 + cytotoxic T cells in the spleens of aged mice. Curcumin has a proven safety profile, and hence, can be used to treat aged donors to boost the functionality of their HSCs and also to improve the immunological profile of aged individuals. These data could have implications in various other regenerative medicine protocols as well., (© 2021 John Wiley & Sons Ltd.)

Background: The kinetics of hematopoietic recovery after autologous stem cell transplantation (ASCT) may be affected by laboratory procedures. The aim of this study was to evaluate the influence of characteristics of the cryopreserved units of peripheral blood stem cells (PBSC) on postthawing cell viability and engraftment outcomes after ASCT., Study Design and Methods: This was a retrospective cohort study including individuals referred for ASCT. Cryopreservation was conducted at a single processing facility between 2014 and 2019, and patients received clinical care at six transplant centers. Covariates and outcome data were retrieved from participants' records., Results: The study population comprised 619 patients (345 [55.7%] male). Median age was 53 years. Multiple myeloma was the most common diagnosis (62.7%). Higher preapheresis CD34+ cell count, lower nucleated cell (NC) concentration per cryobag, and composition of the cryoprotectant solution (5% dimethyl sulfoxide [DMSO] and 6% hydroxyethyl starch) were statistically significantly associated with higher postthawing cell viability. The linear regression model for time to neutrophil and platelet engraftment included the infused CD34+ cell dose and the composition of the cryoprotectant solution. Patients who had PBSC cryopreserved using 10% DMSO solution presented six times higher odds (odds ratio [OR] = 6.9; 95% confidence interval [CI]: 2.2-21.1; p = .001) of delayed neutrophil engraftment (>14 days) and two times higher odds (OR = 2.3, 95%CI: 1.4-3.7; p = .001) of prolonged hospitalization (>18 days)., Discussion: The study showed that mobilization efficacy, NC concentration, and the composition of the cryoprotectant solution significantly affected postthawing cell viability. In addition, the composition of the cryoprotectant solution significantly impacted engraftment outcomes and time of hospitalization after ASCT., (© 2021 AABB.)

Background: Bone marrow graft cell content impacts engraftment potential after allogeneic hematopoietic cell transplantation (alloHCT). Surrogates, such as intraoperative total nucleated cell count (ioTNC), are of unclear utility in predicting final graft characteristics. In addition, demographic and clinical factors may influence graft cellular profile and recipient engraftment., Procedure: We retrospectively reviewed marrow harvests at our institution performed between 2009 and 2019. During this time, an ioTNC was measured after 50% of the projected final graft volume was collected. Regression models were used to assess associations between ioTNC (cells/µL) and final graft CD34+ (cells/mL), and between graft and donor characteristics and final graft CD34+ (cells/mL)., Results: Fifty-three marrow harvests and donor-recipient pairs were analyzed. Median (range) donor and recipient ages were 13 (0.7-28) years and 9 (0.2-21) years, respectively. The median ratio of donor/recipient weight was 1.225 kg (range 0.31-7.13). Median total volume of harvested marrow was 15.3 mL/kg (range 4.3-20.4) of donor weight and 19.4 mL/kg (range 4.7-87.4) of recipient weight. Median ioTNC was 20 930/µL (range 6600-44310) or 2.1 × 10 9 /mL, corresponding to median predicted final graft TNC of 3.59 × 10 8 /kg recipient weight (range 1.28-19.42 × 10 8 ). Simple linear regression between ioTNC and CD34+ cells/mL resulted in an R 2 of 0.42. Least absolute shrinkage and selection operator (LASSO) regression produced a moderately predictive model consisting of ioTNC, donor age, and donor weight (adjusted R 2  = 0.7) of final graft CD34+ cells/mL., Conclusions: ioTNC and certain donor characteristics correlate moderately well with marrow product CD34+ cells/mL, potentially informing donor selection and marrow procurement strategies., (© 2021 Wiley Periodicals LLC.)

Accumulative radiation exposure leads to hematopoietic or tissue aging. Whether hematopoietic stem cells (HSCs) are involved in lung damage repair in response to radiation remains controversial. The aim of this study is to identify if HSC can transdifferentiate to pneumonocytes for radiation-induced damage repair. To this end, HSCs from male Rosa mT/mG mice were isolated by fluorescence-activated cell sorting (FACS) and transplanted into lethally irradiated female CD45.1 mice. 4 months after transplantation, transplanted HSC was shown to repair the radiation-induced tissue damage, and donor-derived tdTomato (phycoerythrin, PE) red fluorescence cells and Ddx3y representing Y chromosome were detected exclusively in female recipient lung epithelial and endothelial cells. Co-localization of donor-derived cells and recipient lung tissue cells were observed by laser confocal microscopy and image flow cytometry. Furthermore, the results showed HSC transplantation replenished radiation-induced lung HSC depletion and the PE positive repaired lung epithelial cells were identified as donor HSC origin. The above data suggest that donor HSC may migrate to the injured lung of the recipient and some of them can be transdifferentiated to pneumonocytes to repair the injury caused by radiation.