Hematopoietic Stem Cells and Their Niche in Bone Marrow.

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Neutrophil generation from hematopoietic progenitor cells and induced pluripotent stem cells (iPSCs): potential applications.

Neutrophils are the most frequent immune cell type in peripheral blood, performing an essential role against pathogens. People with neutrophil deficiencies are susceptible to deadly infections, highlighting the importance of generating these cells in host immunity. Neutrophils can be generated from hematopoietic progenitor cells (HPCs) and embryonic stem cells (ESCs) using a cocktail of cytokines. In addition, induced pluripotent stem cells (iPSCs) can be differentiated into various functional cell types, including neutrophils. iPSCs can be derived from differentiated cells, such as skin and blood cells, by reprogramming them to a pluripotent state. Neutrophil generation from iPSCs involves a multistep process that can be performed through feeder cell-dependent and feeder cell-independent manners. Various cytokines and growth factors, in particular, stem cell facto, IL-3, thrombopoietin and granulocyte colony-stimulating factor (G-CSF), are used in both methods, especially, G-CSF which induces the final differentiation of neutrophils in the granulocyte lineage. iPSC-derived neutrophils have been used as a valuable tool for studying rare genetic disorders affecting neutrophils. The iPSC-derived neutrophils can also be used for disease modeling, infection research and drug discovery. However, several challenges must be overcome before iPSC-derived neutrophils can be used therapeutically in transplantation medicine. This review provides an overview of the commonly employed protocols for generating neutrophils from HPCs, ESCs and iPSCs and discusses the potential applications of the generated cells in research and medicine.

In vitro generation of murine myeloid-derived suppressor cells from hematopoietic progenitor cells.

One of the hallmarks of cancer is the expansion and accumulation of highly immunosuppressive myeloid cells known as myeloid-derived suppressor cells (MDSCs). To study MDSCs biology, differentiation from hematopoietic progenitor cells (HPC) is an useful tool to elucidate the biological and biochemical mechanisms associated with acquisition of immune suppressive activity and expansion in cancer. Although this is one of the protocols performed to study immune suppressive myeloid cells, differentiation of MDSCs from HPC is a method that allows to modify conditions of the supernatants used. In this protocol, we outline the process of differentiating HPCs into MDSCs in vitro using tumor explant supernatants to recapitulate the tumor microenvironment.

In vitro differentiation of human CD45+ CD34+ induced hematopoietic progenitor cells from iPSCs using a monolayer system.

Hematopoietic stem cells (HSCs) represent crucial target cells in the management of hematopoietic and immune system disorders. Unfortunately, the primary source of hematopoietic stem cells is limited. Hematopoietic stem cells derived from induced pluripotent stem cells (iPSCs) hold great promise for applications in cell therapy, disease modeling, and drug screening. To achieve a consistent induction method, one specific induction scheme capable of reliably generating CD34 and CD45 double-positive cells from iPSCs was optimized, employing a comparative analysis and screening of various induction methods. The comprehensive induction procedures are outlined in this document.

Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood.

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.

Technology of genomic balancing of chromatin of autologous hematopoietic stem cells for gene therapy of fatal immune-mediated diseases of civilization, extended life expectancy and sudden human death prevention.

A biotechnology for personalized ex vivo gene therapy based on molecular genomic balancing of hematopoietic stem cell (HSC) chromatin with nucleosome monomers of human genomic DNA (hDNAnmr) has been developed and implemented in the clinic to change (to "correct") mutant chromosome loci genomes of dominant HSC clones that form mono- and oligoclonal hematopoiesis during aging and major (oncological, cardiovascular, neurodegenerative and autoimmune) fatal immune-mediated diseases of civilization. A fundamentally new biotechnological approach has been applied to the delivery of genetic material into eukaryotic stem and progenitor cells by establishing an artificial "recombinogenic situation" in them to induce homologous recombination (equivalent replacement) of mutant DNA regions with healthy hDNAnmr. In experimental preclinical trials, the effectiveness of genomic balancing technology has been proven to reduce the risk of sudden death in old animals and to increase the lifespan of outbred mice by 30% and Wistar rats by 57%. The improvement in their quality of life, compared with the control, is explained by an increase in the telomeric regions of the HSCs and HPCs chromosomes by 1.5-2 times. The potential of the technology to slow down the hereditary neurodegenerative diseases on the model of amyotrophic lateral sclerosis is shown. The effectiveness of this technology in clinical practice is presented on the example of a terminal patient with stage 4 neuroendocrine cancer. This technology used in the treatment of a number of oncological, neurodegenerative, autoimmune and hereditary diseases with clonal hematopoiesis is able to arrest the progression of the disease, prevent its recurrence, prolong the active life of a person, increase the average life expectancy and prevent sudden death.

Phenotypical changes of hematopoietic stem and progenitor cells in COVID-19 patients: Correlation with disease status.

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play a crucial role in the context of viral infections and their associated diseases. The link between HSCs and HPCs and disease status in COVID-19 patients is largely unknown. This study aimed to monitor the kinetics and contributions of HSCs and HPCs in severe and non-severe COVID-19 patients and to evaluate their diagnostic performance in differentiating between healthy and COVID-19 patients as well as severe and non-severe cases. Peripheral blood (PB) samples were collected from 48 COVID-19 patients, 16 recovered, and 27 healthy controls and subjected to deep flow cytometric analysis to determine HSCs and progenitor cells. Their diagnostic value and correlation with C-reactive protein (CRP), D-dimer, and ferritin levels were determined. The percentages of HSCs and common myeloid progenitors (CMPs) declined significantly, while the percentage of multipotent progenitors (MPPs) increased significantly in COVID-19 patients. There were no significant differences in the percentages of megakaryocyte-erythroid progenitors (MEPs) and granulocyte-macrophage progenitors (GMPs) between all groups. Severe COVID-19 patients had a significantly low percentage of HSCs, CMPs, and GMPs compared to non-severe cases. Contrarily, the levels of CRP, D-dimer, and ferritin increased significantly in severe COVID-19 patients. MPPs and CMPs showed excellent diagnostic performance in distinguishing COVID-19 patients from healthy controls and severe from non-severe COVID-19 patients, respectively. Collectively, our study indicated that hematopoietic stem and progenitor cells are significantly altered by COVID-19 and could be used as therapeutic targets and diagnostic biomarkers for severe COVID-19.

[Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation].

OBJECTIVE: To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation. METHODS: Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed. RESULTS: Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05). CONCLUSION: Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

A machine-learning model that incorporates CD45 surface expression predicts hematopoietic progenitor cell recovery after freeze-thaw.

BACKGROUND AIMS: Sufficient doses of viable CD34+ (vCD34) hematopoietic progenitor cells (HPCs) are crucial for engraftment. Additional-day apheresis collections can compensate for potential loss during cryopreservation but incur high cost and additional risk. To aid predicting such losses for clinical decision support, we developed a machine-learning model using variables obtainable on the day of collection. METHODS: In total, 370 consecutive autologous HPCs, apheresis-collected since 2014 at the Children's Hospital of Philadelphia, were retrospectively reviewed. Flow cytometry was used to assess vCD34% on fresh products and thawed quality control vials. The ratio of vCD34% thawed to fresh, which we call "post-thaw index," was used as an outcome measure, with a "poor" post-thaw index defined as <70%. HPC CD45 normalized mean fluorescence intensity (MFI) was calculated by dividing CD45 MFI of HPCs to the CD45 MFI of lymphocytes in the same sample. We trained XGBoost, k-nearest neighbor and random forest models for the prediction and calibrated the best model to minimize falsely-reassuring predictions. RESULTS: In total, 63 of 370 (17%) products had a poor post-thaw index. The best model was XGBoost, with an area under the receiver operator curve of 0.83 evaluated on an independent test data set. The most important predictor for a poor post-thaw index was the HPC CD45 normalized MFI. Transplants after 2015, based on the lower of the two vCD34% values, showed faster engraftment than older transplants, which were based on fresh vCD34% only (average 10.6 vs 11.7 days, P = 0.0006). CONCLUSIONS: Transplants taking into account post-thaw vCD34% improved engraftment time in our patients; however, it came at the cost of unnecessary multi-day collections. The results from applying our predictive algorithm retrospectively to our data suggest that more than one-third of additional-day collections could have been avoided. Our investigation also identified CD45 nMFI as a novel marker for assessing HPC health post-thaw.

The Role of Inflammation in Age-Associated Changes in Red Blood System.

Aging-related anemia contributes to frailty syndrome, cognitive decline and early mortality. The study aim was to evaluate inflammaging in relation to anemia as a prognostic indicator in affected older patients. The participants (73.0 ± 7.2 years) were allocated into anemic (n = 47) and non-anemic (n = 66) groups. The hematological variables RBC, MCV, MCH, RDW, iron and ferritin were significantly lower, whereas erythropoietin EPO and transferrin Tf tended toward higher values in the anemic group. Approx. 26% of individuals demonstrated transferrin saturation TfS < 20%, which clearly indicates age-related iron deficiency. The cut-off values for pro-inflammatory cytokine IL-1ß, TNFα and hepcidin were 5.3 ng/mL, 97.7 ng/mL and 9.4 ng/mL, respectively. High IL-1ß negatively affected Hb concentration (rs = -0.581, p < 0.0001). Relatively high odds ratios were observed for IL-1ß (OR = 72.374, 95%Cl 19.688-354.366) and peripheral blood mononuclear cells CD34 (OR = 3.264, 95%Cl 1.263-8.747) and CD38 (OR = 4.398, 95%Cl 1.701-11.906), which together indicates a higher probability of developing anemia. The results endorse the interplay between inflammatory status and iron metabolism and demonstrated a high usefulness of IL-1ß in identification of the underlying causes of anemia, while CD34 and CD38 appeared useful in compensatory response assessment and, in the longer term, as part of a comprehensive approach to anemia monitoring in older adults.

In Vitro Generation of Murine Dendritic Cells from Hoxb8-Immortalized Hematopoietic Progenitors.

Mouse dendritic cells (DCs) are routinely generated based on cells isolated form the bone marrow (BM) and cultured in the presence of growth factors that support DC development, such as FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Guo et al., J Immunol Methods 432:24-29, 2016). In response to these growth factors, DC progenitors expand and differentiate, while other cell types die during the in vitro culture period, ultimately leading to relatively homogenous DC populations. An alternative method, which is discussed in detail in this chapter, relies on conditional immortalization of progenitor cells with DC potential in vitro using an estrogen-regulated form of Hoxb8 (ERHBD-Hoxb8). Such progenitors are established by retroviral transduction of largely unseparated BM cells with a retroviral vector expressing ERHBD-Hoxb8. Treatment of ERHBD-Hoxb8-expressing progenitors with estrogen results in Hoxb8 activation, which blocks cell differentiation and allows for expansion of homogenous progenitor cell populations in the presence of FLT3L. These cells, referred to as Hoxb8-FL cells, retain lineage potential for lymphocyte and myeloid lineages, including the DC lineage. Upon removal of estrogen (inactivation of Hoxb8), Hoxb8-FL cells differentiate into highly homogenous DC populations in the presence of GM-CSF or FLT3L akin to their endogenous counterparts. Given their unlimited proliferative capacity and amenability for genetic manipulation, for example, by CRISPR/Cas9, these cells provide a large number of options to investigate DC biology. Here, I am describing the method to establish Hoxb8-FL cells from mouse BM, as well as procedures for DC generation and gene deletion using lentivirally delivered CRISPR/Cas9.

Shaping immunity for life: Layered development of CD8+ T cells.

Historically, the immune system was believed to develop along a linear axis of maturity from fetal life to adulthood. Now, it is clear that distinct layers of immune cells are generated from unique waves of hematopoietic progenitors during different windows of development. This model, known as the layered immune model, has provided a useful framework for understanding why distinct lineages of B cells and γδ T cells arise in succession and display unique functions in adulthood. However, the layered immune model has not been applied to CD8+ T cells, which are still often viewed as a uniform population of cells belonging to the same lineage, with functional differences between cells arising from environmental factors encountered during infection. Recent studies have challenged this idea, demonstrating that not all CD8+ T cells are created equally and that the functions of individual CD8+ T cells in adults are linked to when they were created in the host. In this review, we discuss the accumulating evidence suggesting there are distinct ontogenetic subpopulations of CD8+ T cells and propose that the layered immune model be extended to the CD8+ T cell compartment.

Experimental Models of Mouse and Human Hematopoietic Stem Cell Transplantation.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time points post-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6 × BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

Chronic Injury of Mice Bone Marrow Multipotent Hematopoietic Progenitor Cells Induced by Ionizing Radiation / 中国实验血液学杂志

OBJECTIVE@#To explore the chronic injury and its possible mechanism of ionizing radiation on multipotent hematopoietic progenitor cells (MPPs) by determining the related indicators of MPPs in bone marrow of mice post-radiation.@*METHODS@#Sixteen C57BL/6 adult mice were randomly divided into normal control and irradiation groups, 8 mice in each group. The mice in irradiation group were exposed to 6 Gy X-ray. The proportion of bone marrow MPPs, their apoptosis and proliferation 2 months after irradiation were detected by flow cytometry. Mitochondrial activity and levels of reactive oxygen species (ROS) in each MPPs population were detected by Mitotracker Red and DCFDA probes, and the senescent state of MPPs in the bone marrow was analyzed.@*RESULTS@#Ionizing radiation could reduce the proportion of MPPs in mouse bone marrow. The proportions and numbers of MPP1, MPP3 and MPP4 in the bone marrow were significantly decreased after whole-body irradiation with 6 Gy X-ray (P<0.05). In addition, radiation significantly reduced the colony-forming capacity of MPPs in bone marrow (P<0.05), the proportions of apoptotic cells in the MPP1 and MPP4 cell populations increased significantly in the bone marrow (P<0.05). The activity of mitochondria was significantly reduced in the bone marrow MPP2, MPP3 and MPP4 cell populations compared with that of the control group (P<0.05). It was also found that the radiation could significantly increase the ROS levels of MPPs in bone marrow, and the content of ROS in the MPP2, MPP3 and MPP4 cell population of the bone marrow was significantly increased(P<0.05). The senescent cells ratios of MPP1, MPP3 and MPP4 cells in the bone marrow after irradiation were significantly higher than those in the control group (P<0.05).@*CONCLUSION@#Ionizing radiation can cause chronic MPPs damage in mice, which is closely associated with persistent oxidative stress, cells apoptosis, and cellular senescence.

Human Embryonic Stem Cells as a Model for Hematopoietic Stem Cell Differentiation and Viral Infection.

Pluripotent human embryonic stem cell (hESC) lines are a valuable in vitro tool to differentiate specific cell lineages, including cells from all three germ layers, i.e., neuronal cells, myocytes, and hematopoietic cells, including progenitors (described here), lymphoid cells, and myeloid cells. However, dramatically different cell subtypes and functional properties of specific cells can arise depending on the differentiation technique used. We previously optimized hematopoietic stem cell differentiation from two different NIH-approved hESC lines to generate CD34+ hematopoietic progenitor cells (HPCs). Infection of these HPCs with a common herpesvirus (human cytomegalovirus) results in maintenance of viral latency, capability of viral reactivation, recapitulation of viral mutant phenotypes, and virus-induced myelosuppression of hematopoietic differentiation. However, different HPC subpopulations support different viral latency and reactivation phenotypes, and different hESC-to-HPC differentiation methods alter the ratio of stem cell subsets. In addition, differences in differentiation methods are dependent on both protocol/reagents and user techniques. Here, we report a simplified and optimized method to generate large numbers of CD34+ HPCs with consistent phenotypes and demonstrate a comparison of several common methods that can be used to control the ratio of available HPC subpopulations. A key aspect of this approach is that we achieve consistency in differentiation across users in different laboratories and, importantly, among newly trained individuals. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance of human embryonic stem cells (hESCs) Basic Protocol 2: Differentiation of hESCs to hematopoietic progenitor cells (HPCs) Basic Protocol 3: Downstream functional differentiation of hESC-derived HPCs to mature lineages Support Protocol 1: Freezing and testing frozen batches of hESCs Support Protocol 2: Counting hESCs Support Protocol 3: Phenotyping by flow cytometry.

ANALYSIS OF THE INFLUENCE OF PROLONGED IRRADIATION ON HEMATOPOIETIC PROGENITOR CELLS IN GEL DIFFUSION CHAMBERS USING MATHEMATICAL MODELLING. / ANALIZ VPLYVU PROLONGOVANOGO OPROMINENNIa NA GEMOPOETYChNI KLITYNY-POPEREDNYKY U GELEVYKh DYFUZIINYKh KAMERAKh ZA DOPOMOGOIu MATEMATYChNOGO MODELIuVANNIa.

OBJECTIVE: determining of the functional activity of mice bone marrow hematopoietic progenitor cells, cultivated in gel diffusion chambers, on the stages of hematopoiesis recovery after their prolonged irradiation in the lethal dose in a comparative aspect with the method of colony forming in spleen using mathematical model. MATERIALS AND METHODS: The method of cell cultivation in gel diffusion chambers, cytological methods, mathematical modeling, and statistical methods of research were used. Bone marrow samples extracted from the femur of mice irradiated with a total dose of 8 Gy with a power 0.0028 Gy/min were cultivated in diffusion chambers with semi solid agar in the abdominal cavity of CBA recipient mice. RESULTS: Comparative analysis of the colonyforming efficiency of progenitor cells (CFU) was carried out during cultivation in gel diffusion chambers in the process of hematopoiesis recovery for 30 days, as well as in the spleen of lethally irradiated animals, in accordance with the mathematical model. Analysis of colony forming kinetics in gel diffusion chambers after prolonged exposure to ionizing radiation indicated the biphasic nature of hematopoiesis recovery. Thus, in the first few days after the irradiation a drop in the number of CFU is observed compared to the control, which continues until the 9th day. Subsequently there is a sharp increase in the number of CFU in cell culture, which continues until the complete recovery of hematopoiesis. The obtained data, recalculated per mouse femur, correspond to the results of colony forming in the spleen of irradiated animals, described by K. S. Chertkov and taken as a basis while developing our mathematical model, as well as to its parameters, which describe the process of hematopoiesis recovery. CONCLUSIONS: Conformity of the indices obtained during the cultivation using the method of gel diffusion chambers of mice bone marrow prolongedly irradiated at a total dose of 8 Gy with a power 0.0028 Gy/min, to the results of colony forming in spleen of lethally irradiated mice, which were the basis for mathematical model development, is the evidence of the feasibility of using a mathematical model to assess the process of hematopoiesis recovery by progenitor cells of different maturation levels, and the experimental approach of CFU growing in gel diffusion chambers can be considered as an additional method of researching the hematopoiesis recovery along with the spleen colony method.

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

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

Approaches towards Elucidating the Metabolic Program of Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem cells (HSCs) in bone marrow continuously supply a large number of blood cells throughout life in collaboration with hematopoietic progenitor cells (HPCs). HSCs and HPCs are thought to regulate and utilize intracellular metabolic programs to obtain metabolites, such as adenosine triphosphate (ATP), which is necessary for various cellular functions. Metabolites not only provide stem/progenitor cells with nutrients for ATP and building block generation but are also utilized for protein modification and epigenetic regulation to maintain cellular characteristics. In recent years, the metabolic programs of tissue stem/progenitor cells and their underlying molecular mechanisms have been elucidated using a variety of metabolic analysis methods. In this review, we first present the advantages and disadvantages of the current approaches applicable to the metabolic analysis of tissue stem/progenitor cells, including HSCs and HPCs. In the second half, we discuss the characteristics and regulatory mechanisms of HSC metabolism, including the decoupling of ATP production by glycolysis and mitochondria. These technologies and findings have the potential to advance stem cell biology and engineering from a metabolic perspective and to establish therapeutic approaches.

Non-cryopreserved peripheral blood stem cells as a safe and effective alternative for autologous transplantation in multiple myeloma.

BACKGROUND: Autologous stem cell transplantation is the standard procedure for multiple myeloma and the grafts are usually cryopreserved. Previous studies reported advantages in the use of fresh peripheral blood stem cells (PBSC) autotransplantation compared to cryopreservation of the grafts. This study compared the transplant-related outcomes of two graft preservation methods: fresh storage (4°C/72 h) and cryopreservation (-80°C). STUDY DESIGN AND METHODS: We performed an analysis of 45 patients with multiple myeloma under autotransplantation (17 fresh and 28 cryopreserved) from 2017 to 2021. Fresh PBSC were maintained in the refrigerator for three days in a concentration up to 300 × 103 TNC/µL. Cryopreserved PBSC were concentrated by plasma reduction after centrifugation (950 g/10 min/4°C) and an equal volume of cryoprotection solution was added for a final concentration of 300 × 103 TNC/µL, 5% DMSO, 6% hydroxyethyl starch, and 3% human albumin. RESULTS: Neutrophil engraftment was significantly faster with fresh PBSCs (10 vs. 11.5 days, p = 0.045). Adverse effects were more common in cryopreserved PBSC transplantation (75% vs. 35.3% patients; p = 0.013). Post transplantation hospital stay was 20 and 22 days for fresh and cryopreserved PBSCs respectively (p = 0.091). There was no difference in platelet engraftment time (10.5 days for both; p = 0.133), number of antibiotics used after transplantation (3 for fresh and 2.5 for cryopreserved; p = 0.828), days of antibiotic use after transplantation (12.2 days for fresh and 13.3 days for cryopreserved, p = 0.579), and overall survival (p = 0.736). CONCLUSION: The infusion of fresh PBSC refrigerated for up to three days is effective and safe for autologous transplantation in patients with multiple myeloma, which is a useful alternative to cryopreserved PBSC.