Peripheral Blood Hematopoietic Progenitor Cells Research Articles

How adoptive transfer of components of the donor immune system boosts GvL and prevents GvHD in HLA-haploidentical hematopoietic transplantation for acute leukemia.

Why a new Perspective in allogeneic hematopoietic transplantation? A summary. Nowadays, for high-risk acute leukemia patients without an HLA-matched donor (sibling or volunteer), hematopoietic transplants that use HLA-haploidentical grafts combined with enhanced post transplant immune suppression (i.e., high-dose cyclophosphamide) are widely used. They are associated with low TRM rates. However, they are also associated with significant chronic GvHD while they only partially abrogate leukemia relapse rates. One may speculate that post-transplant immune suppression, required for GvHD prophylaxis, weakens the anti-leukemic potential of the graft. Historically, haploidentical transplants became feasible for the first time through transplantation of T cell-depleted peripheral blood hematopoietic progenitor cells. Lack of post-transplant immune suppression allowed the emergence of donor-versus-recipient NK-cell alloreactions that eradicated AML. In an attempt to improve these results we recently combined an age-adapted, irradiation-based conditioning regimen with transplant of T-cell-depleted grafts and infusion of regulatory and conventional T cells, without any post transplant immune suppression. With the obvious limitations of a single center experience, this protocol resulted in extremely low relapse and chronic GvHD rates and, consequently, in a remarkable 75% chronic GvHD/relapse-free survival in over 50 AML patients up to the age of 65 many of whom at high risk of relapse.

Multiomics Characterization of Normal CD34+ Hematopoietic Progenitor Cells (HPC) and Blasts in the Bone Marrow (BM) and Peripheral Blood (PB) of Patients with Acute Myeloid Leukemia (AML)

BACKGROUNG: Multiparameter flow cytometry (MFC) is traditionally used to asses MRD in AML. However, its sensitivity and specificity lag behind other methods and this precludes a broader application in routine practice. It could be hypothesized the emerging multiomics such as single-cell RNA and antibody sequencing could uncover new differences from normal and leukemia associated immunophenotypes (DfN/LAIP). In addition, the identification of DfN/LAIP may be easier in PB when compared to the BM due to the scarcity of CD34+ HPC in the former sample. AIM: Uncover new DfN/LAIP for improved MFC discrimination between normal CD34+ HPC and blasts in the BM and PB of AML patients. METHODS: A total of 47 samples were analyzed in this study. Namely, paired BM and PB from 5 young healthy adults (20-23yo) and 5 PB samples from older healthy adults (47-88yo); paired BM and PB of 11 newly diagnosed AML (44-79yo) patients, and paired BM and PB collected in 5 of the 11 AML patients at the time of MRD assessment. We used fluorescence activated cell sorting (FACS) to isolate CD34+ HPC from the BM and PB of healthy adults as well as blasts from AML patients. scCITE-Seq with a panel of 600 genes and 50 oligo-conjugated antibodies, selected according to their role in stem cell identification, lineage differentiation, clonal hematopoiesis and aberrant expression in myeloid neoplasms was performed. After quality control filtering, a total of 111,334 cells were analyzed and differential expression of genes and antigens was considered if an adjusted P-value <.05 and a log 2FoldChange (FC) >|1| were observed. RESULTS: We first analyzed the phenotype of paired BM and PB 32,637 CD34+ HPC from younger healthy adults. There were 82 deregulated genes, 50 overexpressed in BM and 32 in PB. CLEC12A, CEBPA and HBB were amongst the top overexpressed genes in BM, whereas CRHBP, ADGRG6 and MEIS1 showed the highest upregulation in PB. These results suggest partial transcriptional overlap between BM and PB CD34+ HPC, with the former showing reduced differentiation. There were no differences in antigen expression. Next, we analyzed the potential effect of aging in the phenotype of 34,102 CD34+ HPC from the PB of young and older healthy adults. There were 34 genes differentially expressed including BCORL1, CBLB, HRAS and KDM6A. These findings might reflect an upregulation of genes associated with clonal hematopoiesis. Furthermore, there were 13 antigens differentially expressed including CD9, CD10, CD33, CD38, CD62L and CD371. The comparison of 56,387 blasts in paired BM and PB samples from newly diagnosed AML patients showed near overlap with only HBB being differentially expressed. There were no differences in antigen expression. We then investigated the extent of phenotypic differences between normal CD34+ HPC vs blasts in the BM and PB of healthy adult's vs AML patients. There were 224 and 15 differentially expressed genes between BM cell types, whereas in PB the numbers were 182 and 11, respectively. The markers that contributed the most in the discrimination between CD34+ HPC vs blasts in PB were WT1, ADM, HMOX1, DDIT3, HOPX, CD371, CD45RA, CD133 and CD47. The overlap between DfN/LAIP identified in PB and BM was 31%. Four of the five patients monitored after treatment were MRD positive in BM using MFC. Of note, BM and PB CD34+ HPC isolated from the five AML patients (including one MRD negative in BM) clustered separately of normal BM and PB CD34+ HPC from healthy adults. There was near overlap between BM and PB cells from AML patients. The markers that contributed the most in the discrimination between PB CD34+ HPCfrom healthy adult's vs resistant cells from AML patients were CXCL16, LILRA5, MME, CD274, CTSL, MINPP1 and AHSP. CONCLUSIONS: Normal CD34+ HPC in PB showed partial overlap with the BM counterpart, with a few phenotypic differences probably related to less differentiation and the effects of aging and clonal hematopoiesis. The phenotype of BM and PB blasts from newly diagnosed AML patients nearly overlapped, which indicates that DfN/LAIP may be equally applicable in BM and PB. Accordingly, the markers included in the scRNA/Ab-seq panel effectively discriminated CD34+ HPC from blasts regardless of the sample of origin. Furthermore, we showed phenotypic divergence between PB CD34+ HPC of healthy adults' vs AML patients after treatment. Taken together, this study identified new DfN/LAIP markers for improved assessment of MRD using MFC in the PB of AML patients.

Adverse events related to central venous catheters (CVC) and the influence of CVC characteristics on peripheral blood hematopoietic progenitor cell collection in children.

The use of peripheral blood progenitor cells (PBPCs) as a source for hematopoietic stem cell transplantation (HSCT) in pediatric healthy donors is still under debate. The risk of a central venous catheter (CVC) placement and catheter-related complications continue to be the main arguments to discourage its use. we present a retrospective analysis of 140 PBPC collections in pediatric patients and donors, describing adverse events (AE) related to CVCs as well as the influence of catheterrelated variables on the efficiency of the leukapheresis. 14 CVC-related AEs were recorded (10%). The most common was fever in 5 patients, 4 of which had a catheter-related bacteriemia. Thrombotic events were only observed in 3 patients with active malignancy. A healthy donor presented a moderate bleeding after catheter withdrawal that resolved with local measures, and none of the rest presented any AE. Regarding variables related to the development of AEs, the subject group (patient or donor) was the only one significantly associated (p < 0.0001). Of interest, efficiency was also related to catheter location, being worse in those located in the femoral vein than in into the jugular or the subclavian veins (p < 0.05). In a multivariate analysis, the only variable significantly associated was catheter size (beta 0.238, p < 0.01). Placing a CVC for PBPC collection in pediatric subjects is overall safe; CVC-related complications in pediatric healthy donors are very rare. Furthermore, we should try to place catheters of the largest caliber possible, since the efficiency of the collection is related to this variable.

Ethylene oxide-type hypersensitivity reactions in G-CSF mobilized, peripheral blood hematopoietic progenitor cell donors and review.

Ethylene oxide (EtO) is a volatile, ringed toxic ether used to sterilize heat-labile plastics including apheresis sets. In the 1980s, EtO-associated severe hypersensitivity reactions during hemodialysis led to widespread adoption of alternative sterilization for dialysis kits but not apheresis tubing sets. We now report several cases of EtO-type hypersensitivity reactions in autologous donors undergoing hematopoietic progenitor cell collection (HPCC). A 10-year retrospective review of allergic EtO-type reactions in adults undergoing HPCC on the COBE Spectra and SPECTRA Optia was performed. Donor medical history and absolute eosinophil counts were compared between cases and 34 HPCC controls. Published EtO reactions during extracorporeal procedures were reviewed with statistical analysis. Graphics and statistics were performed using commercial software. Three autologous HPCC donors experienced EtO-type reactions within 15 min of initiating HPCC, for a 10-year incident rate of 0.08% per procedure and 0.18% per donor. All three reactions occurred using the Spectra Optia and IDL tubing set, for an Optia/IDL specific rate of 0.2% per procedure and 0.5% per donor. There was no correlation between EtO reactions, eosinophil counts, or saline prime dwell times. No patient had classic predisposing risk factors for EtO hypersensitivity. Two patients required medical intervention whereas the third responded by pausing the procedure and slowing the inlet rate. EtO-type hypersensitivity reactions can be observed during HPCC, especially with the Optia IDL tubing set. EtO reactions may be missed due to their rarity and staff unfamiliarity with this clinical entity.

233 - Impact of the COVID-19 Pandemic on Unrelated Allogeneic Hematopoietic Peripheral Blood Progenitor Cell Collections in Canada

233 - Impact of the COVID-19 Pandemic on Unrelated Allogeneic Hematopoietic Peripheral Blood Progenitor Cell Collections in Canada

The Sysmex XN series hematopoietic progenitor cell (XN-HPC) as a predictive marker of stem cell enumeration and products: a systemic review and meta-analysis

ABSTRACT Objectives: Sysmex® XN series hematopoietic progenitor cell (XN-HPC) is a sensitive, fast, and economic analytical method for predicting the yields of peripheral blood stem cell enumeration and products, and does not require a sophisticated or expensive workflow. However, various studies have shown that the characteristics of its diagnostic performance were non-uniform. Methods: We performed a systematic inquiry using PubMed, Embase, and Cochrane Library, to comprehensively search for studies published before November 21, 2021. The pooled specificity (SPE), sensitivity (SEN), negative likelihood ratio (NLR), positive likelihood ratio (PLR), diagnostic odds ratio (DOR) and receiver operating characteristic (ROC) curves were summarized to appraise the diagnostic merit of XN-HPC. A forest plot was used to research the sensitivities and specificities of XN-HPC performance. Subgroup analysis was performed to investigate heterogeneity where of importance. Results: Our research included four studies that assessed the diagnostic performance of XN-HPC in hematopoietic progenitor cell collection. The pooled accuracy was 95.4% (95% CI, 94.3–96.3), SPE was 0.81 (95% CI, 0.71–0.88), SEN was 0.95 (95% CI, 0.75-0.99), NLR was 0.06 (95% CI, 0.01–0.37), PLR was 5.0 (95% CI, 3.0–8.5), DOR was 78 (95% CI, 9–707) and the summary of the area under the ROC was 0.90 (95% CI, 0.87–0.92). Forest plot of sensitivities and specificities from XN-HPC test accuracy studies indicated the existence of high heterogeneity. We deduced that the patients were the source of heterogeneity via subgroup analysis. Conclusions: XN-HPC is an excellent diagnostic marker for quantitative detection of peripheral blood hematopoietic progenitor cells.

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Long-Acting, Oxidation-Resistant IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Wild-Type IL-33, G-CSF or AMD3100

▪Mobilization and collection of peripheral blood hematopoietic stem and progenitor cells (HSPC) has become increasingly important for hematopoietic cell transplantation. Granulocyte-colony stimulating factor (G-CSF) alone or in combination with the CXCR4-inhibitor AMD3100 are the most common mobilization approaches. These are however limited since they (a) fail to mobilize a sufficient number of cells in a significant fraction of patients, and (b) are biased towards myeloid immune reconstitution. A novel, single drug mobilization agent that results in a more balanced - myeloid and lymphoid - reconstitution would therefore be highly favorable to improve transplantation outcome.Interleukin (IL)-33 has recently been described as a potent mobilizer of HSPCs with long-term reconstitution potential. IL-33 is an intracellular protein that is believed to be enzymatically activated, released upon cell death, and inactivated upon oxidation. Whereas mobilization of HSPCs by G-CSF has been linked, at least in-part, to modulating the CXCR4-CXCL12 axis, the mobilizing function of IL-33 has been attributed to a different chemokine, CCL7.Here we asked whether IL-33 mobilizes HSPCs better than current clinical mobilizing agents, G-CSF and/or AMD3100, and how the quality of the mobilized cells compares. By flow cytometry and colony-forming assays, we observed for the first time that IL-33 treatment in mice mobilized Lin- Sca-1+ c-kit+ (LSK) cells and common myeloid progenitors (CMP/CFU-GEMMs) more efficiently than G-CSF or AMD3100 alone, and that IL-33 acts synergistically with both drugs. Next we engineered IL-33 variants lacking native cysteine residues, thereby rendering these variants resistant to oxidative inactivation. These variants had equal or better in vitro activity (a) in cell-based reporter assays and (b) in inducing CCL7 protein production by primary human umbilical vein endothelial cells (HUVECs). In vivo, these variants mobilized LSK cells and, interestingly also common lymphoid progenitor (CLP) and endothelial progenitor cells, more efficiently than wild-type IL-33 or G-CSF. We then engineered a one-armed IL-33-Fc fusion molecule comprising a single oxidation-resistant IL-33 fused to an Fc domain. Interestingly, a single dose of this oxidation-resistant IL-33-Fc variant was sufficient to significantly increase the mobilization of LSK, CLP, and endothelial progenitor cells, whereas oxidation-resistant IL-33 or G-CSF were not.In conclusion, our findings suggest that a single dose of long-acting, oxidation-resistant IL-33 may allow efficient mobilization of high-quality HSPCs including CLP cells, and may thus represent a novel mechanism to improve bone marrow transplantation.All authors were employed by Bayer at the time the studies were conducted. DisclosuresAlt:Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Schopies:Bayer: Employment. Liu:Global Blood Therapeutics: Employment. Kauser:Global Blood Therapeutics: Employment, Equity Ownership. Bringmann:Cerus Corporation: Employment. Thompson:Bayer: Employment. Yuan:Bayer: Employment.

Usefulness of hematopoietic progenitor cell monitoring to predict autologous peripheral blood stem cell harvest timing: A single-center retrospective study

IntroductionIn autologous peripheral blood stem cell harvest (APBSCH), CD34-positive cells have been measured to assess the numbers of hematopoietic stem cells, but measurement requires specialized equipment. Recently, there was a report that peripheral blood hematopoietic progenitor cells (HPCs) are useful indicators of the presence of hematopoietic stem cells. We examined the usefulness of HPC monitoring to predict APBSCH timing. MethodsWe retrospectively analyzed the relationship between HPC and collected CD34-positive cells in 84 consecutive patients who underwent APBSCH. ResultsAccording to the receiver operating characteristics curve for the collection of ≥2 × 106 CD34-positive cells/kg, the HPC cut-off value on the day before collection was 21/μL, while that on the day of collection was 41/μL. No significant factors were found in the univariate analysis except for the HPC count on the day before collection (p < 0.001) and the day of collection (p < 0.001). According to the multivariate analysis, the HPC count on the day before collection (p < 0.001) and the day of collection (p < 0.001) were also factors that strongly influenced the quantity of CD34-positive cells collected. ConclusionOur results suggest that the HPC count on not only the day of collection but also the day before collection is a good indicator for appropriate APBSCH timing.

Multiplex CRISPR/Cas9 Genome Editing Targeting the BCL11A/HBG Axis Maximizes Fetal Hemoglobin Reinduction but Generates Chromosomal Translocations Which Persist In Vivo

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means &amp;lt;0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Collection and Processing of Mobilized Mouse Peripheral Blood at Lowered Oxygen Tension Yields Enhanced Numbers of Hematopoietic Stem Cells.

Mobilized peripheral blood (mPB) hematopoietic stem (HSCs) and progenitor (HPCs) cells are primary sources for hematopoietic cell transplantation (HCT). Successful HCT requires threshold numbers of high-quality HSCs to reconstitute hematopoiesis long-term. Nevertheless, considerable percentages of patients and healthy donors fail to achieve required thresholds of HSCs with current mobilization regimens. In this present study we demonstrate that similar to mouse bone marrow (BM) and human cord blood, collection and processing of mouse Granulocyte Colony Stimulating Factor (G-CSF)-, AMD3100/Plerixafor- or G-CSF plus AMD3100/Plerixafor-mobilized HSCs in 3% O2 results in enhanced numbers of rigorously-defined phenotypic and for G-CSF - and G-CSF plus AMD3100/Plerixafor - mPB enhanced functionally-engrafting HSCs. These results may be of potential clinical utility. Graphical Abstract.

Correlation of Various Methods of Hematopoietic Progenitor Cell (HPC) Estimation with Standard Flowcytometric CD34 Enumeration

Background and Objectives: Enumeration of Hematopoietic Progenitor Cell (HPC) is vital to decide the Time To Initiate Harvest (TTIH) and Adequacy Of Harvest Dose (AOHD). Standard of care used for HPC enumeration is flowcytometric CD34+ enumeration, but it is expensive, time-consuming and requires skilled staff to perform the test. Alternatively, HPC-count by advanced automated cell analyzer is cheaper, quicker and easy-to-perform test. Our objective was to find correlation of HPC count with CD34+ enumeration in leukapheresis. Materials and Methods: An observational, prospective study was conducted in the year 2018-2019. A total of 126 samples were included in the study, the peripheral blood group comprised of 42 samples and apheresis group of 84 samples. The samples were simultaneously tested for CD34+ expression and complete blood count (CBC) which included the HPC count, WBC count, MNC count and IG (Immature Granulocyte) index and correlation analysis was performed with CD34 + flowcytometric count. The cut-off of peripheral blood HPC count for the target dose of 5*106 CD34+ cells/kg was established using Receiver Operator Curve. Results: The correlation coefficient (r) of HPC with CD34+ count was 0.617 and 0.699 for peripheral blood and apheresis sample respectively, which was statistically significant. The correlation with MNC, IG and WBC count was not very significant. A cut-off value of PB HPC was established to be 66 HPC/µL with a positive predictive value of 94.12%.The cost of CD34+ flow cytometric enumeration was six times that of HPC enumeration by analyzer. Conclusion: The HPC count is a cheaper, rapid and easy test and can be clinically applied to predict TTIH. Funding Statement: None. Declaration of Interests: None. Ethics Approval Statement: The study was approved by Institutional Review Board. (MICR-849/2018).

Modulation of the HGF/c-Met Axis Impacts Prolonged Hematopoietic Progenitor Mobilization Following Trauma and Chronic Stress.

Trauma and hemorrhagic shock trigger mobilization of hematopoietic progenitor cells (HPC) from bone marrow to peripheral blood. Hepatocyte growth factor (HGF), tyrosine-protein kinase Met (c-Met), matrix metallopeptidase 9 (MMP-9), and corticosterone regulate this mobilization process. We hypothesized that beta-blockade with propranolol and sympathetic outflow inhibition with clonidine following trauma and chronic stress would decrease hematopoietic progenitor cell mobilization. Sprague-Dawley rats were randomized to undergo three models of injury and stress: lung contusion, LC plus hemorrhagic shock (LCHS), or LCHS plus chronic restraint stress for 2 h daily (LCHS/CS). Propranolol and clonidine were administered by daily intraperitoneal injection until sacrifice on day seven. Bone marrow HGF, c-Met, and MMP-9 were measured by real-time PCR. Plasma corticosterone was measured by ELISA. Percentage HPC in peripheral blood was measured by flow cytometry. Propranolol and clonidine significantly decreased bone marrow MMP-9 expression, plasma corticosterone levels, and HPC mobilization, and significantly increased hemoglobin levels. HPC mobilization was greatest following LCHS/CS (5.4 ± 1.8) and was significantly decreased by propranolol (2.2 ± 0.9, P < 0.001) and clonidine (1.7 ± 0.5, P < 0.001). Hemoglobin (g/dL) was lowest following LCHS/CS (12.3 ± 1.2) and was significantly increased by propranolol (13.7 ± 0.4, P = 0.022) and clonidine (14.1 ± 1.1, P < 0.001). Severe injury was associated with increased bone marrow HGF, c-Met, and MMP-9, circulating corticosterone, HPC mobilization, and persistent anemia. Attenuating the neuroendocrine response to injury and stress with propranolol and clonidine reduced MMP-9 expression, corticosterone levels, HPC mobilization, and the degree of anemia.

Smoking is an important factor that affects peripheral blood progenitor cells yield in healthy male donors.

Smoking could reduce the CD34+ cells in peripheral blood of healthy individual. This study aimed to investigate the correlation between smoking load and the effect of peripheral blood hematopoietic progenitor cells (PBPCs) mobilization by granulocyte colony-stimulating factor (G-CSF) alone in healthy donors. Retrospective analysis was performed on 145 healthy adult PBPCs donors who underwent PBPCs mobilization and collection. Smoking factors were evaluated and correlated with mobilization responses, as indicated by the collected CD34+ cells concentration. The collected CD34+ cells concentration was closely related to pre-CD34 (P < .001) and CD34+ cells collected per volume blood processed (P < .001) which suggested that collected CD34+ cells concentration was a reliable indicator of PBPCs mobilization efficiency. The heavy smoking donors revealed significantly lower collected CD34+ cells concentration, compared to that of the nonsmoking (P < .001) and light smoking donors (P < .05). The levels of collected CD34+ cells in light smoking were also obviously lower than that in nonsmoking donors (P < .05).There were no obvious differences in the collected CD34+ cells concentration, overall processed blood volume and total collected CD34+ cells between nonsmoking and smoking cessation groups (P = .490; P = .464; P = .819). Cigarette smoking is an important factor that affects the yield of PBPCs in male donors, especially when the smoking load is more than five pack-years. Mobilization of PBMCs could be restored by smoking cessation in chronic smokers.

Cytokine-induced hematopoietic stem and progenitor cell mobilization: unraveling interactions between stem cells and their niche.

Peripheral blood hematopoietic stem and progenitor cells (HSPCs), mobilized by granulocyte colony‐stimulating factor, are widely used as a source for both autologous and allogeneic stem cell transplantation. The use of mobilized HSPCs has several advantages over traditional bone marrow–derived HSPCs, including a less invasive harvesting process for the donor, higher HSPC yields, and faster hematopoietic reconstitution in the recipient. For years, the mechanisms by which cytokines and other agents mobilize HSPCs from the bone marrow were not fully understood. The field of stem cell mobilization research has advanced significantly over the past decade, with major breakthroughs in the elucidation of the complex mechanisms that underlie stem cell mobilization. In this review, we provide an overview of the events that underlie HSPC mobilization and address the relevant cellular and molecular components of the bone marrow niche. Furthermore, current and future mobilizing agents will be discussed.

Processing of G-CSF Mobilized Peripheral Blood Hematopoietic Progenitor Cells (PB-HPC) from Matched- Related or Unrelated Donors Undergoing CD34 Enrichment with T Cell Addback in Children, Adolescents and Adults with Malignant and Nonmalignant Diseases: Rapid Engraftment, Immune Cell Reconstitution and Sustained Donor Chimerism

Sixty to seventy percent of patients undergoing HLA identical transplants will develop grade II-IV acute GVHD, despite using prophylactic immunosuppressants. Several studies have demonstrated that manipulation utilizing ex-vivo CD34+ positively selected grafts result in 4-5 log T-cell depletion with limited T-cell add back follow with early engraftment and decreased aGVHD (Silva et al, Blood Journal, 2015 & Chakrabarti et al, BMT, 2003). We previously demonstrated the safety and efficiency of CD34+ enriched cells with 2 × 105 CD3 (MNC)/kg add back in pediatric SCD patients resulting in rapid engraftment, donor chimerism, and low incidence of aGVHD (Geyer/Cairo et al, BJH, 2012). Utilizing CD34+ cell selection, children, adolescents, and adults with a matched or partially matched family donor or matched unrelated donors allogeneic PB-HPC transplant can safely receive and tolerate with a low incidence of serious (Grade III/IV) acute and chronic GVHD. Patients received CD34+ enriched products processed by using the CliniMACS CD34+ Reagent System (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD34+ enriched HPC product was either a fresh infusion or cryopreserved and stored until time of transplantation. A target of 5 × 106 CD34+enriched/kg recipient weight with a T cell add back of 2 × 105 CD3+/kg (MNC) was infused. Sixteen patients underwent BMT with median age of 55 years (23 months to 71 years). Patients' disease status was as follows: complete remission (CR) 2 in four AML patients, CR1 in 9 AML patients, Lymphohistiocytosis (n=1), Macrophage Activating Syndrome (n=1), CR2 in one Non-Hodgkins Lymphoma patient. Nine patients received 10/10 HLA-matched unrelated donor BMT, two 9/10 HLA-matched unrelated donors, five had 6/6 HLA-matched sibling transplant. PB-HPC products contained 0.59 × 1010 CD3/kg (±0.23), and 6.77 × 106 CD34/kg (±1.27). After CD34 enrichment, the PB-HPC CD34% recovery was 73.1±3.08% with a log T cell depletion of 4.9 (±0.11). The target transplant dose per patient was 5 × 106 CD34 enriched/kg. Sixteen and patients had myeloid and 13 patients had platelet engraftment with a median of 11.5 days and 16 days, respectively (Fig. 1a and Fig. 1b). Two patients died prior to platelet engraftment one due to multi-organ system failure following septic shock and the other patient due to refractory AML. One patient died after myeloid and platelet engraftment secondary to disease progression. Early and late peripheral blood chimerism were ≥ 98.4% at 19.9 days (±12) and 92.5% at 180 days (n=4). The probability of grade II-IV aGVHD was 20% (CI95: 0.1-66.9) (Fig 2). One patient had grade II skin aGVHD and one patient had grade II gut aGVHD. No patient had cGVHD. This study demonstrates the safety and efficiency of CD34+ enriched PB-HPC products with T-cell addback in AlloSCT recipients with a low probability of acute GVHD.

Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy.We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture.By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between.After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC.Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO).To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status.As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2weeks after MI. In female Lewis inbred rats (n=9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponinI concentrations 24h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day0) and 14days after MI or sham surgery (day14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P>0.05) in both groups at day14, without interaction between groups. In the intervention group, higher day0 CFU-GM counts showed a relationship to lower fractional shortening on day14 (ρ=-0.82; P<0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14days after MI with circulating CFU-GM counts on day0 might imply functional colony count as a predictive factor for outcome after infarction.

Performance and safety of femoral central venous catheters in pediatric autologous peripheral blood stem cell collection.

Autologous peripheral blood hematopoietic progenitor cell collection (A-HPCC) in children typically requires placement of a central venous catheter (CVC) for venous access. There is scant published data regarding the performance and safety of femoral CVCs in pediatric A-HPCC. Seven-year, retrospective study of A-HPCC in pediatric patients collected between 2009 and January 2017. Inclusion criteria were an age ≤ 21 years and A-HPCC using a femoral CVC for venous access. Femoral CVC performance was examined by CD34 collection rate, inlet rate, collection efficiency (MNC-FE, CD34-FE), bleeding, flow-related adverse events (AE), CVC removal, and product sterility testing. Statistical analysis and graphing were performed with commercial software. A total of 75/119 (63%) pediatric patients (median age 3 years) met study criteria. Only 16% of children required a CVC for ≥ 3 days. The CD34 collect rate and CD34-FE was stable over time whereas MNC-FE decreased after day 4 in 80% of patients. CD34-FE and MNC-FE showed inter- and intra-patient variability over time and appeared sensitive to plerixafor administration. Femoral CVC showed fewer flow-related AE compared to thoracic CVC, especially in pediatric patients (6.7% vs. 37%, P = 0.0005; OR = 0.12 (95%CI: 0.03-0.45). CVC removal was uneventful in 73/75 (97%) patients with hemostasis achieved after 20-30 min of pressure. In a 10-year period, there were no instances of product contamination associated with femoral CVC colonization. Femoral CVC are safe and effective for A-HPCC in young pediatric patients. Femoral CVC performance was maintained over several days with few flow-related alarms when compared to thoracic CVCs.

332 - Familial Haploidentical (FHI) T-Cell Depleted (TCD) with T-Cell Addback Stem Cell Transplantation for Patients with High-Risk Sickle Cell Disease (SCD) (IND 14359)

Allogeneic stem cell transplantation (AlloSCT) from HLA-matched unaffected sibling donors (MSD) has been successful for high-risk SCD, and is the only known curative therapy (Freed/Cairo et al, BMT, 2012). We have recently demonstrated 100% event free survival and absence of sickle cell symptoms following reduced toxicity conditioning and HLA matched sibling bone marrow or cord blood AlloSCT (Bhatia/Cairo et al, BMT, 2014). However, 5 out of 6 children who might benefit from this therapy lack an HLA matched family donor. Identifiable matched unrelated adult donors (URD) in this ethnic group are extremely limited and results from unrelated cord blood transplants are poor (Radhakrishnan K/Cairo et al., BBMT 2013, Kamani et al., BBMT, 2012). We previously demonstrated the use of positive CD34 selection followed by T cell add back (2 x 105 CD3/kg) from unrelated donors in pediatric recipients with both malignant and nonmalignant disease lead to 100% engraftment with minimal acute GVHD (aGVHD). In a high-risk FHI TCD thalassemia study, 16/22 cases engrafted without aGVHD and with 90% overall survival (Sodani et al., Blood, 2010). FHI TCD AlloSCT could expand the donor pool and improve outcomes for patients with high risk SCD. This SCD consortium trial is investigating the safety, feasibility, EFS, donor chimerism, graft failure, aGVHD and chronic GVHD (cGVHD), and infectious mortality after FHI TCD AlloSCT in high-risk SCD patients ([Figure 1][1]). High risk features included one or more of the following: ≥1 CVA, ≥2 ACS, ≥3 VOC in past 2 years, or 2 abnormal TCDs. Patients (2-20.99 yrs) without an 8/8 HLA MSD or URD and who have ≥1 high-risk SCD features were eligible. Patients received hydroxyurea 60 mg/kg/d and azathioprine 3mg/kg/d, day -59 – day -11, fludarabine (30mg/m2/d x5d), busulfan (3.2 mg/kg/d x4d [<4yrs of age: 4 mg/kg/d x4d]), thiotepa (10 mg/kg/d x1d), cyclophosphamide (50mg/kg/d x4d), R-ATG (2mg/kg/d x4d), and TLI (500cGy) followed by FHI T-cell depleted AlloSCT. AGVHD prophylaxis included tacrolimus single agent. We utilized the CliniMACS (IND 14359) to enrich for peripheral blood hematopoietic progenitor cells (HPC's); target dose of 10 x 106 CD34+ cells/kg with 2 x 105 CD3+ T cells/kg added back as a final CD3/kg concentration. Six patients have received AlloSCT to date ([Figure 2][2]). All patients utilized maternal donors who encountered no complications during collection. All had early neutrophil engraftment (median day +9), ≥98% whole blood chimerism and ≥85% RBC donor chimerism, no aGVHD or cGVHD ([Figure 2][2]). One patient developed late hepatic SOS and died at day +59; the remainder are alive and free of disease (day +12 to +642). One more patient has been enrolled and has begun conditioning and others are in the early part of their transplant process. Early results indicate FHI TCD AlloSCT is feasible in high-risk SCD patients who lack a MSD or URD. A larger cohort with longer term follow-up is needed to assess long-term safety and outcomes (Supported by FDA 5R01FD004090 and a grant from Otsuka) (IND #14359 and NCT 01461837). http://www.sicklecelltransplantconsortium.org ![Figure 1][3] Figure 1 ![Figure 2][3] Figure 2 Disclosures No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: pending:yes