Hematopoietic Stem 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.

Autophagy regulates the maturation of hematopoietic precursors in the embryo

An understanding of the mechanisms regulating embryonic hematopoietic stem cell (HSC) development would facilitate their regeneration. The aorta-gonad-mesonephros region is the site for HSC production from hemogenic endothelial cells (HEC). While several distinct regulators are involved in this process, it is not yet known whether macroautophagy (autophagy) plays a role in hematopoiesis in the pre-liver stage. Here, we show that different states of autophagy exist in hematopoietic precursors and correlate with hematopoietic potential based on the LC3-RFP-EGFP mouse model. Deficiency of autophagy-related gene 5 (Atg5) specifically in endothelial cells disrupts endothelial to hematopoietic transition (EHT), by blocking the autophagic process. Using combined approaches, including single-cell RNA-sequencing (scRNA-seq), we have confirmed that Atg5 deletion interrupts developmental temporal order of EHT to further affect the pre-HSC I maturation, and that autophagy influences hemogenic potential of HEC and the formation of pre-HSC I likely via the nucleolin pathway. These findings demonstrate a role for autophagy in the formation/maturation of hematopoietic precursors.

Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies

Background aimsCryopreservation 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. MethodsAll 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. ResultsA 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. ConclusionsReducing 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 registrationPROSPERO; registration number CRD42023476809 registered November 8, 2023.

Quantitative association between gene expression and blood cell production of individual hematopoietic stem cells in mice.

Individual hematopoietic stem cells (HSCs) produce different amounts of blood cells upon transplantation. Taking advantage of the intercellular variation, we developed an experimental and bioinformatic approach to evaluating the quantitative association between gene expression and blood cell production across individual HSCs. We found that most genes associated with blood production exhibit the association only at some levels of blood production. By mapping gene expression with blood production, we identified four distinct patterns of their quantitative association. Some genes consistently correlate with blood production over a range of levels or across all levels, and these genes are found to regulate lymphoid but not myeloid production. Other genes exhibit one or more clear peaks of association. Genes with overlapping peaks are found to be coexpressed in other tissues and share similar molecular functions and regulatory motifs. By dissecting intercellular variations, our findings revealed four quantitative association patterns that reflect distinct dose-response molecular mechanisms modulating the blood cell production of HSCs.

Thrombopoietin treats erythropoietin resistance by correcting EPO-induced progenitorcell depletion

Recombinant human erythropoietin (rHuEPO) is a prevalent treatment for anemia in patients with chronic kidney disease. However, up to 10% of these patients exhibit EPO resistance or hyporesponsiveness, which may be caused by the depletion of erythroid progenitor cells. Thrombopoietin (TPO) has the potential to promote the growth of early progenitor cells and correct the depletion. In this study, we investigate the efficacy and the underlying mechanism of the combination therapy of TPO and EPO to EPO resistance. First, the in vivo studies suggested that intensive EPO treatment induced progenitor cell depletion in the bone marrow, where the depletion was corrected by TPO. Then, colony assays showed that EPO and TPO synergistically enhanced the burst-forming unit-erythroid (BFU-E) production but antagonistically boosted the colony-forming units of megakaryocytes (CFU-MK) production. Also, we found TPO promoted hematopoietic stem and progenitor cells (HSPCs) production, while EPO drove HSPCs toward the erythroid lineage. Additionally, EPO induced more megakaryocytic-erythroid progenitors (MEPs) toward the erythroid output. Model-based simulations indicate the efficacy of this combination therapy for treating EPO-resistant anemia in rats. In conclusion, our study demonstrated the efficacy of combination therapy in addressing EPO-resistant anemia by correcting EPO-induced erythroid progenitor depletion.

Zinc chloride may regulate hematopoietic stem cell aging and pro-inflammatory cytokines in systemic lupus erythematosus

Background: The immune cells of patients with systemic lupus erythematosus (SLE) age earlier than those of normal subjects. However, the senescence of circulating hematopoietic stem cells (HSCs) in patients with SLE is not well understood, and it is unclear whether zinc treatment can regulate the senescence and proinflammatory cytokine production of HSCs in these patients. Methods: Clinical data were collected on 38 patients with SLE and 35 healthy controls (HCs), and the complete blood count, circulating HSC number, and p16 (a senescence marker) expression in the peripheral blood of these participants were analyzed via flow cytometry. Pooled circulating HSCs were isolated using leukapheresis. The effects of zinc chloride exposure on the pooled HSCs of each group were determined in vitro. Levels of the proinflammatory cytokines IL-6 and IL17, regulatory cytokine TGF-β, p16, and regulator T-cells (Tregs) were evaluated 72 h after incubation with 50 or 100 µM zinc chloride. Results: The number of circulating HSCs did not differ between the two groups (p=0.1685). The expression of p16 in HSCs was higher in the SLE group than in the HC group (p = 0.0043), and patients with SLE exhibited higher levels of IL-6, IL-17, and p16 in pooled HSCs (p =0.0025, p<0.0001, and p = 0.0003, respectively), although TGF-β levels did not differ between the groups (p=0.9816). Zinc chloride reduced IL-6, TGF-β, IL-17, and p16 expression in patients with SLE toward HC levels. Treg frequency in pooled HSCs was comparable between the groups (p=0.3997), although a 100 µM zinc chloride treatment significantly depleted the Treg population of patients with SLE (p=0.0001). Conclusions: Circulating HSCs in SLE are more aged and produce more proinflammatory cytokines. Zinc chloride treatment might prevent immunoaging and inhibit proinflammatory cytokine–producing cells in patients with SLE.

Trends in hematopoietic stem cell utilization and cost of cryopreservation

Abstract Autologous stem cell transplantation (ASCT) is the standard of care treatment for multiple myeloma. Standard ASCT procedures collect hematopoietic stem cells (HPSC) in excess for multiple transplants due to reports of high mobilization failure and unsuccessful ASCT collections following myeloablative treatments. Cost analysis studies estimate that one year of cryopreservation and one day of HPSC collection would each cost $5000 per patient. Limited data is available on the usage of the second transplant and the storage costs of non-transplanted products. This study evaluates the utilization of cryopreserved HPSC products and calculates the cost of cryopreservation. A retrospective chart review study of a cohort of 240 deceased MM patients, who received ASCT between January 2010 to December 2015. Demographic and clinical data were obtained from the laboratory and electronic medical record. The cohort was categorized into two groups, with 130 patients in Group 1 (≤60 years) and 110 patients in Group 2 (>60 years). Cumulative incidence of a second transplant was computed from the life table using the Kaplan Meier method. The cost of cryopreservation was calculated based on the time HPSC products remained in the freezers before disposal. PBSC was mobilized using Granulocyte Colony Stimulating Factor (GCSF), or GCSF and Mozobil. The average age of patients at first collection is 61 (IQR: 54-66) years and the average age of death is 64 (IQR: 58 -70) years. The average life span of patients after receiving the first transplant is 3 (IQR: 2-5) years. The average number of collection days is 2 (IQR: 1-3). 65 patients had 1-day collections, 105 had 2-day collections and 70 had ≥3-day collections. 175 patients (73%) had >1 day collection. Only 22 patients (9%) received a second transplant and 18 of them (82%) were in Group 1 (≤60). The cumulative incidence of a second transplant by 5 years is 23% for Group 1 and 4% for Group 2. Two-transplant collections required 492 days of apheresis with an overall cost of $2,500,000; however, one-transplant collections would cost $1,400,000 for 279 apheresis days. The median cost of cryopreservation for each patient who did not receive the second transplant is $15,000 (IQR: $8000 - $25,000). Therefore, the estimated cost for all patients in the study is $3,600,000 until the disposal of HPSC products. The incidence of a second transplant is low across all patients and significantly lower in patients greater than 60 years of age. Cryopreserved stem cell products remain under-utilized, leading to substantial financial burden. The duration of HPSC collection and cryopreservation must be re-evaluated for future treatment plans. Institutional policies should be revised based on the utilization trend to lower the operational cost of storage.

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.

API-192, an Allogeneic CD19/20 CAR-NKT Cell Product Derived from Cord Blood CD34+ HSPCs for the Treatment of B-Cell Malignancies

Autologous chimeric antigen receptor (CAR)-T cell therapies induce durable remissions and improve survival in patients with advanced B cell malignancies. However, challenges such as manufacturing variability, cost, and time to treatment highlight the need for an allogeneic, off-the-shelf approach that preserves the efficacy of autologous CAR-T. Additionally, antigen escape is one of the established mechanisms of resistance to current CAR-T therapies which target a single antigen. Classical natural killer T (NKT) cells express an invariant T cell receptor (iTCR) that is restricted to the conserved CD1d protein, making it a promising cell type for allogeneic therapies as they do not require TCR gene editing to avoid graft-vs-host (GvH) disease. Furthermore, NKT cells mediate dendritic cell cross-priming, target immunosuppressive myeloid cells/macrophages in the tumor microenvironment, and directly kill CD1d + tumor cells through the iTCR. Endogenous NKT cells are an extremely rare population in the blood (<1% of lymphocytes), posing manufacturing challenges. API-192 is a human cord blood CD34 + hematopoietic stem and progenitor cell-derived NKT cell product engineered to express dual CARs targeting CD19 and CD20 and armored with soluble IL-15 for improved expansion and persistence. The Appia Cells Utilized for Allogeneic (ACUA) platform enables the production of large numbers of product NKT cells co-expressing multiple transgenes at high purity (>90% iTCR + 19CAR + 20CAR +) with no purification step or gene editing. Phenotypically, API-192 cells are predominantly CD4 -CD8 - or CD4 -CD8 +, express major activating NK receptors, and harbor intracellular stores of cytotoxic molecules, resembling endogenous NKT cells. API-192 kills Raji and Nalm6 tumor cells in a CAR-specific and dose-dependent manner and can control multiple rounds of tumor challenge in vitro. API-192 secretes pro-inflammatory cytokines and proliferates in response to tumor antigen . In human tumor xenograft models in immunocompromised mice, API-192 robustly expands, suppresses tumor growth, and exhibits prolonged persistence, enabling control of a tumor rechallenge 70 days after initial dosing. API-192 expresses lower levels of MHC I and MHC II molecules relative to conventional CAR-T cells and elicits less IFN-γ release by NK and T cells in mixed lymphocyte reaction (MLR) assays, indicative of a more favorable immunogenic profile. Compared to conventional CAR-T cells, API-192 also secretes minimal levels of IFN-γ in response to co-culture with healthy donor peripheral blood mononuclear cells, consistent with the expression of a non-HLA restricted TCR and low GvH risk. These results demonstrate the feasibility, potency, and potential safety of API-192, warranting further development as a novel allogeneic therapy.

MYELODYSPLASTIC SYNDROMES

Introduction: Myelodysplastic syndrome is a pathology characterized by its hematopoietic, pluripotential ineffective process in the bone marrow, i.e. causing dysplasia in at least one of its hematopoietic lines with functional and morphological alterations, which, at some point may evolve into leukemia. Objective: review, analyze and describe the most notable features of myelodysplastic syndromes. Methodology: A total of 20 articles were analyzed in this review, including review and original articles, as well as clinical cases, of which 11 bibliographies were used because the other articles were not relevant to this study. The sources of information were PubMed, SciELO, Google Scholar, Medigraphic and Cochrane; the terms used to search for information in Spanish, Portuguese and English were: myelodysplastic syndrome, hematopoietic, anemia, leukemia. Results: It is estimated to have an incidence of 4-12 conditions out of 100 000 patients per year, and it can reach up to 30 out of 100 000 in individuals older than 70 years of age. The occurrence of this anomaly in pediatric age and in young adults is rare and infrequent, and a certain predominance in the male sex has been reported, with a ratio of 1:5, and no relationship with race has been found. Most patients develop symptomatology related to cytopenias and anemia; isolated neutropenias and thrombocytopenias also occur, although not less frequently. Conclusions: Myelodysplastic syndromes are highly complex disorders of low incidence. They mainly affect the production of hematopoietic stem cells, being disorders of difficult approach, due to the mutation and the affection that these disorders entail, triggering aggressive dysplastic and blood diseases such as anemias and leukemias, which makes therapeutic decisions difficult, so it is extremely important to make a correct diagnosis based on a clinical, morphological and exclusion analysis. In addition, the patients condition and age must be taken into account, so that the treatment is effective and the patients survival is adequate. Keywords:MDS:Myelodysplastic syndrome.

Facilitators and barriers in oncology clinical practice influencing nurses' completion of GRASP Workload Measurement System to ensure patient-centred care.

Hospital administrators have a great interest in obtaining a valid and reliable nursing workload measurement to help determine the hours of care needed per patient; one such method available is the GRASP Workload Measurement System. At Sunnybrook Health Sciences Centre, nurses' GRASP compliance and accuracy varies and often does not meet the target of 90%. The target assists the organization in estimating ongoing nursing workload and patients' care needs, while ensuring the provision of safe and appropriate care that is fiscally responsible. The objective of the quality initiative reported in this paper was to identify the facilitators and barriers that influence nurses' completion of GRASP. The quality improvement project was conducted using a mixed-method design with a sample of 28 nurses working in oncology acute care and palliative care inpatient units. The Theoretical Domain Framework (TDF), often used in behaviour change studies, was used in designing the questionnaire survey and interview questions that listed pertinent and measurable factors that may influence nurses' GRASP completion. Facilitators included: nurses' knowledge about the role GRASP has in funding and staffing levels, job responsibility, and perception of GRASP as a potential tool to organize work. Barriers identified by nurses included insufficient GRASP knowledge, limited access to workstations and computers, GRASP tool elements not capturing the complexity of the nursing work, time constraints, increased patients' acuity, and care demands. In addition to the Theoretical Domain Framework, the Normalization Process Theory was used to guide the implementation and evaluation of the recommendations to enhance nurses' GRASP compliance and adherence practice.

Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition.

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because patients with GATA2 haploinsufficiency have inborn mutations, prenatal defects are likely to influence disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos remain unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We showed that Gata2+/- HSPCs initiate, but cannot fully activate, hematopoietic programming during EHT. In addition, due to the reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress endothelial identity to complete maturation. Finally, we showed that hematopoietic-specific induction of gfi1b could restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates the pivotal role of Gata2 in the regulation of the transcriptional network governing HSPC identity throughout the EHT.

Reducing Microbial Contamination in Hematopoietic Stem Cell Products and Quality Improvement Strategy: Retrospective Analysis of 1996-2021 Data.

Sterility and safety assurance of hematopoietic stem cell (HSC) products is critical in transplantation. Microbial contamination can lead to product disposal and increases the risk of unsuccessful clinical outcomes. Therefore, it is important to implement and maintain good practice guidelines and regulations for the HSC collection and processing unit in each hospital. We aimed to share our experiences and suggest strategies to improve the quality assurance of HSC processing. We retrospectively analyzed microbial culture results of 11,743 HSC products processed over a 25-year period (January 1996 to May 2021). Because of reorganization of the HSC management system in 2008, the 25-year period was divided into periods 1 (January 1996 to December 2007) and 2 (January 2008 to May 2021). We reviewed all culture results of the HSC products and stored aliquot samples and collected culture results for peripheral blood and catheter samples. Of the 11,743 products in total, 35 (0.3%) were contaminated by microorganisms, including 19 (0.5%) of 3,861 products during period 1 and 16 (0.2%) of 7,882 products during period 2. Penicillium was the most commonly identified microorganism (15.8%) during period 1 and coagulase-negative Staphylococcus was the most commonly identified (31.3%) during period 2. HSC product contamination occurred most often during HSC collection and processing. The contamination rate decreased significantly during period 2, when the HSC management system was reorganized. Our results imply that handling HSC products by trained personnel and adopting established protocols, including quality assurance programs, aid in decreasing the contamination risk.

Optimizing of the isolation of hematopoietic stem cell in bone marrow transplantation in children

Most of collected hematopoietic stem cell (HSCs) products need processing in order to isolate stem cells, squeeze out of plasma and erythrocytes. There are two main aims for bone marrow (BM) enrichment: reduction of immunogenicity of AB0 incompatible transplants and/or preventing toxicity of hemolysis during cryopreservation. In our center we have implemented two methods for BM enrichment: manual technic using 10 % HAES (hydroxyethyl starch) and automatic cell separator. In order to optimize the process, we examined retrospectively the parameters which could have a great impact on final efficiency of engraftment, such as reduction of hematocrit, CD34 + , WBC recovery and cell viability. This study was a retrospective analysis of 46 pediatric patients (pts) who underwent autologous or allogeneic HSCT. There were performed 27 procedures using cell separator and 19 with HAES technique. This study showed that cell separator processing is significantly less damaging for stem cells than widely longer, manual HAES technique. Comparing RBC depletion and WBC recovery both used techniques are same efficient and good enough but we found out a significant difference in the efficiency of CD34 + recovery which was much higher in a technique of cell separator. We examined also the effect of addition of packed red blood cells (PRBCs) to the BM on purifying and efficiency of HSCs isolation. Doing so it decreased only the WBC recovery during sell separator processing. To sum up after series of analyzes we found out that cell separator is more convenient than HAES technique in most of considered aspects. Furthermore, cell separator use is cheaper and needs less time for processing.

Validation of long-term handling and storage conditions for hematopoietic stem cell products for autologous transplants.

Hematopoietic stem cells (HPSCs) are multipotent stem cells that can differentiate into lymphoid and myeloid progenitors, giving rise to white blood cells (WBCs), red blood cells (RBCs), and platelets. HPSCs are a widely used treatment for many hematological non-malignant and malignant disorders. HPSCs can be used in the fresh or cryopreserved state for future use. Fresh HPSCs are typically stored at 2-6°C for up to 72 hours and are primarily used for allogeneic transplants or autologous transplants in myeloma and lymphoma patients. However, in some cases of autologous donations, HPSC transplantation is delayed more than three days after collection. In such situations, the cells are thawed after short-term preservation, resulting in a 35% cell viability loss. This study aimed to investigate the quality of HPSCs products after long-term storage exceeding 72 hours. The quality of HPSCs products was assessed by measuring viable CD34+ cell count, the total number of nucleated cells (TNC), and HPSCs recovery after different storage intervals of up to 120 hours in hypothermal storage. The mean total cell viability decreased by 2.18% within 72 hours and 7.4% within 120 hours, while mean CD34+ cell recovery was 92.61 % at 72 hours and 83.83 % at 120 hours in hypothermal storage. The mean TNC recovery was 89.93% at 72 hours and 76.18 % at 120 hours. All products were free from bacterial contamination for up to 120 hours under hypothermal storage conditions.

Sterility Testing for Hematopoietic Stem Cells.

Over the last two decades, rapid technological advances have led to the wide adoption of cell and gene therapy products for the treatment of a variety of disease states. In this study, we reviewed the literature between 2003 and 2021 to provide a summary of overarching trends associated with microbial contamination in hematopoietic stem cells (HSCs) derived from peripheral blood, bone marrow, and cord blood. We provide a brief background on the regulatory context for human cells, tissues, and cellular and tissue-based products (HCT/Ps) as regulated by the US Food and Drug Administration (FDA), sterility testing expectations for autologous (Section 361) and allogeneic (Section 351) HSC products, and discuss clinical risks associated with the infusion of a contaminated HSC product. Finally, we discuss the expectations for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) for the manufacturing and testing of HSC based on Section 361 and Section 351 categorization, respectively. We provide commentary on what is practiced in the field and discuss the critical need for updates to professional standards that keep pace with advancing technologies with an aim to clarify expectations for manufacturing and testing facilities to improve standardization across institutions.

Cpeb1b-mediated cytoplasmic polyadenylation of shha mRNA modulates zebrafish definitive hematopoiesis

During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog-Vegf-Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.

Evaluating the effectiveness of a training program to support nurses to administer cryopreserved hematopoietic stem cells by intravenous push method.

A training program was developed to prepare registered nurses (RNs) at one cellular therapy centre to administer cryopreserved cells by intravenous (IV) push method. There are two main methods of infusion for dimethyl sulfoxide (DMSO) cryopreserved hematopoietic stem cell (HSC) products, gravity drip and IV push. Administering DMSO by either route can cause hypersensitivity reactions. Administration of HSCs by gravity drip is slower, resulting in fewer DMSO reactions. However, prolonged exposure of DMSO once the cells are thawed increases the risk of cellular damage. The faster IV push method reduces cell damage and decreases staff time. An environmental review within Canadian transplant centres found that in most adult centres, nurses administer by gravity drip, and when IV push is required, cryopreserved hematopoietic stem cells are administered by physicians. Our centre's method was IV push by a physician or nurse practitioner (NP). As transplant numbers grew, capacity to perform this skill needed to expand. To maintain the current benefits of the IV push method and increase capacity in a hematopoietic transplant program, the role of infusing stem cells by the IV push method was transitioned from the NPs and physicians to RNs. A successful training program utilizing simulation to support these oncology nurses in learning the new skill was developed and evaluated.

Lineage tracking to reveal the fate of hematopoietic stem cells influenced by Flk2− multipotent progenitors after transplantation

After transplantation, hematopoietic stem cells (HSCs) sustain blood cell regeneration throughout the patient’s life. Recent studies suggest that several types of mature blood cells provide feedback signals to regulate HSC fate. However, the potential feedback effect of hematopoietic progenitor cells has not been characterized to date. The present investigation demonstrated that multipotent progenitors (MPPs) promoted T cell production of HSCs when both cell types were cotransplanted in mice. Using genetic barcodes to track individual HSCs in mice, we found that the increased T cell production by HSCs was associated with the combined effects of altered lineage bias and clonal expansion during HSC differentiation. We showed that MPP and HSC co-transplantation promoted the multilineage differentiation of HSCs in the short term while preserving lymphoid-specialized HSC differentiation in the long term. Our findings indicate that MPPs derived from HSCs regulate the fate of HSCs after bone marrow transplantation.

Key priorities for cell & gene therapy manufacturing evolution in 2023 & beyond

David McCall, Commissioning Editor,BioInsights, spoke to David DiGiusto, Senior Vice President, Stem Cells and Regenerative Medicine, National ResilienceDavid DiGiusto is the Senior Vice President for Stem Cells and Regenerative Medicine at National Resilience. He has over 28 years of experience in the scientific, clinical and regulatory aspects of cells as therapeutic agents including the isolation, characterization and genetic modification of hematopoietic stem cells, T cells and regenerative medicine products for clinical applications. He has been instrumental in the creation of seven GMP compliant biologics manufacturing facilities and associated quality systems, production and QC testing programs. Under his direction, plasmid DNA, CAR-T cells, regulatory T cells, engineered stem cell grafts and gene modified hematopoietic stem cell products have been manufactured and released for use in Phase 1/2 clinical trials. David is a major contributor to first in human studies for Cancer and HIV Gene Therapy and has developed methods for assessing ex vivo stem cell manipulations using in vitro and in vivo models. David is a former member of the NIH recombinant DNA advisory committee (RAC) and an independent consultant to the Biotechnology industry.