Production Of Hematopoietic Cells Research Articles

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

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

Prospection of Therapeutic Agents Targeting Aurora Kinase, a Protein in the Treatment of Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is characterized by an imbalance in the production and development of hematopoietic lymphoid cells, a malignant disease capable of affecting the proliferation-selection of hematopoietic cells. The Aurora kinase A protein participates in several steps of the mitosis process. Its deregulation can trigger the process of carcinogenesis, which has become a therapeutic target of interest for computational prediction and the development of inhibitory drugs. Studies report its overexpression in malignant cells of patients with ALL. Objective: The present study aims to prospect new molecules to identify a potential inhibitor of Au-rora kinase A for the pharmaceutical market. Methodology: Virtual screening and molecular docking study was performed using the MCULE and DockThor web servers. The pharmacodynamic and pharmacokinetic profile of the molecules were evaluated using the Swiss ADME and ProTox-II programs were used. Results: Ten molecules were identified by virtual screening, in which only two, MCULE-349 and MCULE-796, showed the best score, binding, at the site of action of the protein, interacting positively with amino acids, lipid-soluble molecules with low toxicity and with violation of only 1 Lipinski rule. Conclusion: Both molecules interact with the site of action, acting as inhibitors or blockers of the catalytic site, becoming potential Aurora kinase A inhibitors and anticancer molecules.

PXS5505-MF-101: A Phase 1/2a Study to Evaluate Safety, Pharmacokinetics and Pharmacodynamics of Pxs-5505 in Patients with Primary, Post-Polycythemia Vera or Post-Essential Thrombocythemia Myelofibrosis

Background: Myelofibrosis (MF) is characterized by a progressive increase in extracellular matrix in the bone marrow (BM) associated with decreased production of hematopoietic cells. Though Janus kinase (JAK) inhibitors provide clinical benefit in patients (pts) with MF, they have no direct effect on BM fibrosis (BMF) and thereby limited disease modifying effects. MF is associated with elevated expression of most lysyl oxidase (LOX) isoforms which cross link collagen and elastin. PXS-5505 is a pan-LOX inhibitor that prevents the cross-linking of collagen and elastin and exhibits anti-fibrotic effects in murine models of MF. PXS5505-MF-101 is an ongoing multi-center phase 1/2a study of PXS-5505 in pts with primary, post-polycythemia vera (PV) or post-essential thrombocythemia (ET) MF. (NCT04676529) The study started with two phases: Dose Escalation (DEP) and Cohort Expansion (CEP). The DEP has been completed, establishing a dose of 200mg BID to carry forward into the CEP. During the CEP, up to 24 pts are to be treated for 6 months. Preliminary data from the CEP is presented here. Methods: Study objectives are to determine PXS-5505 safety/tolerability, pharmacokinetics (PK) and pharmacodynamics (PD), BMF changes, spleen volume and MF symptoms (MFSAF). Eligible pts must have primary or post-ET/PV MF of Int-2/high risk; relapsed/refractory or intolerant/ineligible to available JAK inhibitors and have symptomatic disease. Results: At data cutoff (30/Jun/2023), 21 pts have enrolled in the CEP (7 F and 14 M); age 60-86 yrs (median 72 yrs); of these, 10 have completed 24 weeks of Rx and 20 pts have completed ≥1-month Rx. 8 pts had post-ET MF, 5 post-PV MF and 8 PMF; 9 pts have withdrawn from the CEP. Total drug exposure in the CEP is 372 weeks, median 162 days (min 25, max 191). Preliminary CEP PK data are consistent with the DEP with steady state reached by Day 28 with no accumulation observed past this point. The PD data from the DEP demonstrated excellent inhibition of LOX as measured by LOX and LOXL2 target engagement (data previously presented) and this level of inhibition continues to be observed in CEP. [Figure 1] All 9 pts completing 24 weeks PXS-5505 with baseline (BL) biopsies available showed stable reticulin fibrosis at each visit; collagen fibrosis was reduced in 5/9 pts. [Figure 2] Hematological parameters were stable; 7/10 pts had stable/improved hemoglobin and 8/10 had stable/improved platelets over 24 weeks including 3 pts with Grade 4 thrombocytopenia at BL. 4/10 pts had improvements in MF-SAF TSS of ≥20% with 2/10 pts of ≥40% at 24 weeks. No SVR35 responses were seen. There has been 1 fatal AE (febrile neutropenia) on Rx, and 2 post-Rx discontinuation/completion (sepsis, acute myocardial infarction), all unrelated. There have been 6 Rx related adverse events (TRAEs); 2 Grade 1 (vomiting, urticaria), 2 Grade 2 (purpura, hypomagnesaemia), 1 Grade 3 (platelet count decreased), 1 Grade 4 (anemia, noted as leading to drug withdrawal). There have been no serious TRAEs reported. Enrolment into the CEP continues and an additional cohort is planned using PXS-5505 as an add-on to Ruxolitinib Rx. Conclusion: PXS-5505 has been well tolerated with no dose limiting toxicity or serious TRAEs. PD results indicate excellent LOX inhibition at the 200mg BID level. Further, there are preliminary indications of disease modification characterized by stable/improved blood counts and an improvement in collagen fibrosis. These results are encouraging given the poor prognosis seen after Ruxolitinib discontinuation in pts with MF and warrant continued investigation in an earlier Rx setting.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

Frequency of Aplastic Anemia in Children (1-15 years) with New-Onset Pancytopenia

Background: Pancytopenia is a medical disorder in which the usual quantities of red blood cells, white blood cells, and platelets are depleted. This condition can arise from various factors, including diminished production of hematopoietic cells, ineffective hematopoiesis, or the destruction of these cells in the peripheral blood. Objective: To determine the frequency of aplastic anemia in children (1-15 years) with new-onset pancytopenia presenting at Children Hospital Lahore. Study Design: Cross sectional study. Settings: Department of Pediatric Medicine, Children Hospital, Lahore Pakistan. Duration: From August 2022 January 2023. Methods: This research included 250 children, covering both genders, with ages ranging from 1 month to 15 years old. These children had all been diagnosed with pancytopenia within the past month. The primary focus of the study was to determine the prevalence of aplastic anemia within this group of children. This prevalence was then examined in relation to their age, gender, and socioeconomic backgrounds. Results: The patient group had a mean age of 6.74 years, primarily consisting of children aged 1-10 years (86.4%). Gender distribution was fairly balanced, with 55.2% males and 44.8% females. Most patients belonged to the middle class (48.8%), followed by the lower class (35.2%) and high class (16.0%). Pallor was the most common, with 86.36% of patients experiencing it. Fever and bruises/purpura were also frequent, affecting 64.55% and 59.55% of patients, respectively. Aplastic anemia was observed in 33.60% of the children, while 66.4% did not have this condition. Conclusion: In children who had recently developed pancytopenia, the incidence of aplastic anemia was determined to be 33.60%. Interestingly, no noteworthy disparities in the occurrence of aplastic anemia were observed with regard to age, gender, or socioeconomic status.

Pancytopenia as Another Collateral Damage of Covid-19 Infection in a Previously Healthy Child

Aim and Background: Pancytopenia is a reduction of all the three cells of peripheral blood lineage: leukocytes, erythrocytes and platelets. The bone marrow can be primarily hypocellular in pancytopenia either due to reduction of hematopoietic cell production or ineffective haemopoiesis. Generally, infections, toxins or malignant transformation of the marrow are responsible for most of the cases of pancytopenia.1 After the incidence of Covid-19 pandemic, pancytopenia has been seen to occur as a rare complication secondary to SARS-COV 2. Here, we report a unique case of pancytopenia in a previously healthy child with SARS-CoV-2 infection.

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

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

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

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

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

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

Effects of calreticulin mutations on cell transformation and immunity.

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

EVALUATION OF PANCYTOPENIA WITH BONE MARROW INTERPRETATION

Background: Pancytopenia is one of the most common clinico-haematological entity observed in our day to day clinical practice. It is a disorder in which all the three major elements of blood (i.e. red blood cells, white blood cells and platelets) are decreased in number. The causes of pancytopenia may be due to decrease in hematopoietic cell production in the marrow resulting from infections, toxins, malignant cell inltration, post- chemotherapy or post-radiation. Aims and Objectives: 1) To study the etiology and clinical presentation of pancytopenia in all age groups. 2) To correlate with bone marrow interpretation Materials &amp; Methods: This is a prospective study which was conducted among 50 patients of pancytopenia in the Clinical Pathology, Government General Hospital,Kurnool from January 2021 to October 2022 Bone marrow aspiration was done by using Salah's bone marrow puncture needle. Smears were made from bone marrow aspirate (BMA) and stained by Leishman stain and special stains like Perl`s wherever necessary. The smears were assessed for cellularity, differentiation and maturation of erythroid, myeloid and megakaryocytic lineage, M:E ratio, Plasma cells, Lymphocytes and parasites/ abnormal cells. In the present study the commonest cause of Pancytopenia was Megaloblastic anemia (70%) follo Results: wed by Dimorphic anemia (20%). The less common conditions were Multiple Myeloma (6%),Chronic Myeloid Leukemia(2%),Acute Leukemia(2%). The present study concludes that com Interpretation and Conclusion: plete primary hematological investigations along with bone marrow aspiration in pancytopenic patients are helpful for understanding disease process and to diagnose or to the rule out causes of pancytopenia. These are also helpful in planning for further investigations and management.

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

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

Hematopoietic Flt3+ Multipotent Progenitors Are Organized in Discrete Functionally and Hierarchically Distinct Populations

Blood production in the bone marrow (BM) is classically viewed as a constant hematopoietic flux originating from a small pool of self-renewing hematopoietic stem cells (HSCs). In this model, quiescent HSCs regularly differentiate and give rise to a complex set of transient multipotent progenitors (MPPs) that serve as compartments of amplification through division, while undergoing progressive lineage specification Recent studies exploiting sophisticated genetic marking for in vivo cell-fate tracking have challenged this paradigm and suggested that HSCs have a limited contribution to steady state blood production (Sun, Nature 2014, Busch, Nature 2015; Rodriguez-Fraticelli, Nature 2018). The interpretation of these results and the extent of the HSC contribution to native hematopoiesis remains a matter of debate (Pucella, Annu Rev Cell Dev Biol 2020; Rodriguez-Fraticelli, Curr. Opin. Hematol 2021). Beyond this debate, these studies have highlighted the underestimated contribution of the MPP compartment to the daily hematopoietic output in unperturbed conditions. This new understanding raises questions about the functional properties of the MPP compartment in steady state condition and its ability to independently sustain prolonged hematopoietic cell production. Here we revisited the early hematopoietic hierarchy present in the Lin- Kit+ Sca1+ (LSK) fraction that includes all HSC and MPP (MPP1-5) compartments. Highly-multiplexed CITE-Seq single cell analyses with over 60 antibodies identified a novel LSK CD62L- Flt3+ population (CD62L- MPP4) that clusters within the MPP compartments but displays a cell cycle gene signature similar to the one found in quiescent HSCs. Analysis of the Mki67 reporter mouse model and nucleotide analogue incorporation assays directly confirmed the existence of this unexpected cell cycle diversity in MPP4 during unperturbed hematopoiesis. Consistent with the molecular data, these analyses characterized a resting CD62L- and a proliferative CD62L+ MPP4 compartments. These two populations showed similar cloning efficiency in methylcellulose assays indicating that both populations behave as intermediary MPP compartments. When assessed in short-term culture, both populations were responsive to Flt3L stimulation but displayed specific kinetics of division. They also showed distinct short-term reconstitution ability upon transplantation in correlation with their cell cycle activity. Analysis of these populations in native condition using the H2B-GFP label-retaining system further confirmed the specific properties of the resting CD62L- MPP4 cells, distinct from the other proliferative MPP2, MPP3 and CD62L+ MPP4 compartments. Finally, these studies uncovered a novel early path of differentiation that involves the MPP5 cells (LSK Flt3+ CD48- CD150-) sequentially acquiring Flk2 and CD48 expression to generate the resting CD62L- MPP4 compartment, which in turn gives rise to cycling CD62L+ MPP4 cells. Altogether, these results challenge the accepted dichotomy between quiescent HSCs and dividing progenitors. While difference in lineage potential has been historically a major criterium to classify MPP populations, we established a new dimension at the MPP diversity, based on their proliferative activity at steady state. These results particularly highlight a new path of differentiation directly linking MPP5, a quiescent compartment with residual self-renewal capacity, to the resting CD62L- Flt3+ MPP cells. Based on these results, we proposed that slow-dividing Flt3+ MPPs act as a key transitional compartment that can buffer the hematopoietic cell production and therefore regulate hematopoietic homeostasis at steady state. We envision that this novel compartment could be a key intermediary for hematopoietic malignancies driven by the acquisition of Flt3 mutations and a cellular target for intervention.

Sitagliptin Alleviates Excessive Autophagy in Macrophages in Immune Thrombocytopenia Patients to Promote Megakaryopoiesis Via the AMPK/mTOR Pathway

Introduction: The pathogenesis of ITP is complex and mainly manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. Sitagliptin, a DPP-4 inhibitor, was reported to play a key role in the production and engraftment of hematopoietic cells by modulating SDF-1α and several colony-stimulating factors, thus enhancing hematopoietic stem and hematopoietic progenitor cell recovery. However, whether sitagliptin has the potential to promote megakaryocyte maturation and platelet production in ITP is unclear. Therefore, this article explores the effect of macrophage autophagy on megakaryopoiesis in ITP patients and investigates its underlying role in the pathogenesis of ITP in order to explore whether sitagliptin could be a potential treatment for ITP. Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP. Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively. Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Isolation and Characterization of Fetal Liver Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are responsible for the generation and maintenance of pools of multipotent precursors that ultimately give rise to all fully differentiated blood and immune cells. Proper identification and isolation of HSCs for functional analysis has greatly facilitated our understanding of both normal and abnormal adult hematopoiesis. Whereas adult hematopoiesis inmice and humans is driven by quiescent HSCs that reside almost exclusively within the bone marrow (BM), developmental hematopoiesis is characterized by a series of transient progenitors driving waves of increasingly mature hematopoietic cell production that occur across multiple anatomical sites. These waves of hematopoietic cell production are also responsible for the generation of distinct immune cell populations during development that persist into adulthood and contribute uniquely to adult immunity. Therefore, methods to properly isolate and characterize fetal progenitors with high purity across development become increasingly important not only for defining developmental hematopoietic pathways, but also for understanding the contribution of developmental hematopoiesis to the immune system. Here, we describe and discuss methods and considerations for the isolation and characterization of HSCs from the fetal liver, the primary hematopoietic organ during fetal development.

Hoxa9/meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance.

<i>HOXA9</i> and <i>MEIS1</i> are co-expressed in over 50% of acute myeloid leukaemia (AML) and play essential roles in leukaemogenesis, but the mechanisms involved are poorly understood. Diverse animal models offer valuable tools to recapitulate different aspects of AML and link <i>in vitro</i> studies to clinical trials. We generated a double transgenic zebrafish that enables <i>hoxa9</i> overexpression in blood cells under the draculin (<i>drl</i>) regulatory element and an inducible expression of <i>meis1</i> through a heat shock promoter. After induction, Tg(<i>drl</i>:<i>hoxa9</i>;<i>hsp70</i>:<i>meis1</i>) embryos developed a preleukaemic state with reduced myeloid and erythroid differentiation coupled with the poor production of haematopoietic stem cells and myeloid progenitors. Importantly, most adult Tg(<i>drl</i>:<i>hoxa9</i>;<i>hsp70</i>:<i>meis1</i>) fish at 3 months old showed abundant accumulations of immature myeloid precursors, interrupted differentiation and anaemia in the kidney marrow, and infiltration of myeloid precursors in peripheral blood, resembling human AML. Genome-wide transcriptional analysis also confirmed AML transformation by the transgene. Moreover, the dihydroorotate dehydrogenase (DHODH) inhibitor that reduces leukaemogenesis in mammals effectively restored haematopoiesis in Tg(<i>drl</i>:<i>hoxa9</i>;<i>hsp70</i>:<i>meis1</i>) embryos and improved their late survival. Thus, Tg(<i>drl</i>:<i>hoxa9</i>;<i>hsp70</i>:<i>meis1</i>) zebrafish is a rapid-onset high-penetrance AML-like disease model, which provides a novel tool to harness the unique advantages of zebrafish for mechanistic studies and drug screening against <i>HOXA9</i>/<i>MEIS1</i> overexpressed high-risk AML.

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

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

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

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

Flow Cytometry Analysis of Hematopoietic Stem/Progenitor Cells and Mature Blood Cell Subsets in Atherosclerosis.

Advancing age causesphysiologic decline in tissue function. In the hematopoietic system this manifests as a progressive reduction in blood or immune cell function and clonal hematopoiesis, where a mutated hematopoietic stem cell candominate blood cell production andconfer an increased propensity for myeloid malignancy. In the aging cardiovascular system, atherosclerosis causes an inflammatory cell- driven accumulation of lipid-derived plaques in major arteries which constrains blood flow and can lead to myocardial infarction and stroke. Clonal hematopoiesis in the elderly has recently been associated with a substantially increased risk of atherosclerosis-related cardiovascular disease. However, the direct association between deregulated hematopoiesis in clonal hematopoiesis and atherosclerosis is poorly defined. Herein, we describe a flow cytometry method to prospectively analyze the crucial hematopoietic stem/progenitor, inflammatory and lymphoid cell participants in atherosclerosis. This analysis can be applied to decipher the complex relationship between hematopoietic cell types involved in clonal hematopoiesis and atherosclerosis in mouse models.

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

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