Homing Of Hematopoietic Stem Cells Research Articles

Abstract 732: Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia

Abstract Introduction: CXCR4 is a chemokine receptor for CXCL12 that plays a critical role in homing of hematopoietic stem cells (HSCs) to the bone marrow microenvironment. The CXCR4 inhibitor plerixafor is used to increase mobilization of peripheral blood stem cells in combination with filgrastim. This pathway has also been investigated for applications in hematologic malignancies such as acute myeloid leukemia (AML), including clinical trials, with the goal of disrupting AML from the protective marrow environment. These have included use of plerixafor with 7+3 or MEC, BL-8040 with cytarabine, and ulocuplumab with MEC. For the ulocuplumab trial, at the study site of the authors, CR/CRi correlated with >10% CXCR4 expression (Becker et al. ASH 2014). The objective of this preclinical study is to identify the AML patient population most likely to respond to CXCR4 inhibition, and the role of combined beta-adrenergic blockade, as the latter has been shown pre-clinically to augment mobilization of HSCs when combined with a CXCR4 inhibitor, GPC-100 (Sukhtankar et al. PLoS One, 2023). Procedures: AML patient samples (N=124 including 90 blood and 34 bone marrow samples, and 21 with both) were obtained with informed consent on an IRB approved protocol. Multicolor flow cytometry was performed with sequential gating on blasts (by FSC/SSC, CD45/SSC, CD34 or phenotype), and leukemia stem cells (LSCs:CD34+CD38−,CD123+), then analysis for CXCR4 and beta-2 adrenergic receptor (ADRB2) surface expression. Correlations between CXCR4 expression and clinical characteristics including age, gender, new diagnosis vs. relapse, ELN2022 risk, cytogenetics, FLT3/NPM1 mutation status, CR1 duration, and overall survival were performed. GraphPad Prism9 was used for statistical analysis, and Mann Whitney test (two groups) and ordinary one-way ANOVA (>2 groups) were used for comparisons. Results: The range of CXCR4 expression by AML blasts is 0.05 to 99.7% (median 13.1%; mean 16.2%), and by AML LSCs 0 to 99% (median 2%, mean 15.7%). The expression by blasts and LSCs for each patient exhibited tight correlation, R2=0.88 p=1.23 × 10e-25). There was higher % expression of CXCR4 by both blasts and LSCs for new diagnosis, as compared to relapsed, patient samples (p=0.002). There was no correlation between level of CXCR4 expression and the other clinical features. For a subset of 20 patient samples analyzed for both CXCR4 and ADBR2 expression by AML blasts, ADBR2 expression ranged from 0.4 to 20.1%. For the samples that express ADBR2, there was high level expression of CXCR4, range 35.9 to 98.7% (median 78.4%, mean 76%). Conclusion: These studies support further investigation of whether simultaneous blockade of CXCR4 and ADBR2 may potentiate chemotherapy response in AML by limiting microenvironment mediated chemotherapy protection and reveal that patients with new diagnosis may be more susceptible to this approach. Citation Format: Brenda Loera, Chunxiao Zhang, Pina M. Cardarelli, Niña G. Caculitan, Sylvia Chien, Jin Dai, Vivian G. Oehler, Jianying Zhang, Guido Marcucci, Pamela S. Becker. Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 732.

Homing and Engraftment of Hematopoietic Stem Cells Following Transplantation: A Pre-Clinical Perspective.

Hematopoietic stem-cell (HSC) transplantation (HSCT) is used to treat various hematologic disorders. Use of genetically modified mouse models of hematopoietic cell transplantation has been critical in our fundamental understanding of HSC biology and in developing approaches for human patients. Pre-clinical studies in animal models provide insight into the journey of transplanted HSCs from infusion to engraftment in bone-marrow (BM) niches. Various signaling molecules and growth factors secreted by HSCs and the niche microenvironment play critical roles in homing and engraftment of the transplanted cells. The sustained equilibrium of these chemical and biologic factors ensures that engrafted HSCs generate healthy and durable hematopoiesis. Transplanted healthy HSCs compete with residual host cells to repopulate stem-cell niches in the marrow. Stem-cell niches, in particular, can be altered by the effects of previous treatments, aging, and the paracrine effects of leukemic cells, which create inhospitable bone-marrow niches that are unfavorable for healthy hematopoiesis. More work to understand how stem-cell niches can be restored to favor normal hematopoiesis may be key to reducing leukemic relapses following transplant.

A Pilot Study to Determine the Safety and Efficacy of Repeated Hyperbaric Oxygen Therapy in Multiple Myeloma Patients Undergoing High-Dose Therapy and Autologous Stem/Progenitor Cell Transplantation

Background Serum erythropoietin (EPO) negatively impacts hematopoietic stem cell homing and engraftment. Hyperbaric oxygen (HBO) therapy can reduce serum EPO in healthy volunteers and when given prior to stem cell infusion may improve engraftment after hematopoietic stem cell transplant (HSCT). We previously performed a phase II trial randomizing patients with multiple myeloma undergoing high dose melphalan and autologous HSCT 1:1 between a single HBO treatment and no HBO. HBO was given as 100% oxygen, 2.5 ATA for a total of 90 min, in a single treatment on Day 0 prior to the transplant. A total of 99 patients were enrolled with 52 randomized to HBO. HBO significantly reduced EPO levels for the first 24 hours after auto transplant, but EPO levels quickly rebounded by 48 hours after transplant. We hypothesized multiple HBO treatments will keep EPO levels low during the duration of stem cell homing (i.e: the first 72 hours after HSCT) and may improve neutrophil and NK cell recovery. Methods We performed a pilot study to evaluate the safety and tolerability of multiple HBO treatments during autologous HSCT. Patients with multiple myeloma undergoing autologous HSCT were eligible. Patients received HBO therapy, consisting of hyperbaric oxygen at 2.5 ATA for a total of 90 minutes breathing 100% oxygen on days 0 (prior to HSCT), 1, and 2 of transplant. The primary objective of this pilot study was to determine the safety of repeated HBO therapy in the setting, as defined by treatment limiting toxicity (TLT) including occurrence of seizure disorder, pneumothorax, death, or any irreversible grade Ill or any grade IV toxicity within 24 hours of HBO therapy. Secondary objectives were to determine the effects of multiple HBO therapy on neutrophil count recovery and the effect of multiple HBO on serum EPO. Results 10 patients have been enrolled that are evaluable for the primary objective. All patients had multiple myeloma and 4 were female. 9/10 patients successfully completed all 3 HBO treatments and 1/10 completed HBO on days 1 and 2 only because of ear pain on day 0. No patients experienced treatment limiting toxicity. The median times to neutrophil and platelet engraftment was 12 days (range 11-14) and 16 days (range 14-28), respectively. The median length of hospitalization was 13 days (range 12-16). Febrile neutropenia occurred in 3/10 patients with just one incidence of documented infection. This was significantly less than the rate of febrile neutropenia in patients receiving a single treatment of HBO or no HBO (81% and 47%, respectively, p<0.05). 3 patients required red blood cell transfusion and 5 platelet transfusion. Conclusions Multiple HBO treatment were well tolerated without treatment limiting toxicity during autologous HSCT for multiple myeloma.

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

Influence of SPIO labelling on the function of BMSCs in chemokine receptors expression and chemotaxis.

Bone marrow-derived mesenchymal stem cells (BMSCs) are increasingly being used in bone marrow transplantation (BMT) to enable homing of the allogeneic hematopoietic stem cells and suppress acute graft versus host disease (aGVHD). The aim of this study was to optimize the labelling of BMSCs with superparamagnetic iron oxide particles (SPIOs), and evaluate the impact of the SPIOs on the biological characteristics, gene expression profile and chemotaxis function of the BMSCs. The viability and proliferation rates of the SPIO-labeled BMSCs were analyzed by trypan blue staining and CCK-8 assay respectively, and the chemotaxis function was evaluated by the transwell assay. The expression levels of chemokine receptors were measured by RT-PCR and flow cytometry. The SPIOs had no effect on the viability of the BMSCs regardless of the labelling concentration and culture duration. The labelling rate of the cells was higher when cultured for 48 h with the SPIOs. Furthermore, cells labeled with 25µg/ml SPIOs for 48 h had the highest proliferation rates, along with increased expression of chemokine receptor genes and proteins. However, there was no significant difference between the chemotaxis function of the labeled and unlabeled BMSCs. To summarize, labelling BMSCs with 25µg/ml SPIOs for 48h did not affect their biological characteristics and chemotaxis function, which can be of significance for in vivo applications.

Involvement of CXCL12/CXCR4 axis in colorectal cancer: a mini-review.

Migration of metastatic tumor cells is similar to the traffic of leukocytes and has been reported that can be guided by chemokines and their receptors, through the circulation to distant organs. The chemokine CXCL12 and its receptor CXCR4 play an essential role in hematopoietic stem cell homing and the activation of this axis supports malignant events. Binding of CXCL12 to CXCR4 activates signal transduction pathways, with broad effects on chemotaxis, cell proliferation, migration and gene expression. Thus, this axis serves as a bridge for tumor-stromal cell communication, creating a permissive microenvironment for tumor development, survival, angiogenesis and metastasis. Evidence suggests that this axis may be involved in the colorectal cancer (CRC) carcinogenesis. Therefore, we review emerging data and correlations between CXCL12/CXCR4 axis in CRC, the implications for cancer progression and possible therapeutic strategies that exploit this system.

Expanding the Abu Dhabi Bone Marrow Transplant Program (AD-BMT&amp;lt;sup&amp;gt;®&amp;lt;/sup&amp;gt;) Scope with the Collection and Cryopreservation of Cord Blood Stem Cells

<i>Introduction:</i> The umbilical cord blood (UCB) is the blood from the placenta's blood vessels and the portion of the umbilical cord that remains attached to the newborn after the delivery when the cord is clamped and cut. UCB is rich in hematopoietic stem cells (HSC) and other stem cells (SC). Between 1974 and 1988, scientific knowledge and UCB application possibilities for human therapy opened a new significative era in cell therapy, using UCB HSC as an alternative source of SC for bone marrow transplant and the cure of patients with malignant hematological diseases. <i>Objective:</i> The possibility of having a UCB bank (UCB-B) in Abu Dhabi encouraged us to make this analysis. <i>Method:</i> A literature review from Google Scholar, PubMed, SciELO, Scopus, and other sources, about the increasing application of UCB-SC and storage in UCB-B was done to update about all the possibilities for translational medicine. <i>Results:</i> We prepared a UCB overview, including the pioneering use of UCB-SC for HSC transplantation, their biological composition, characteristics, and the UC-associated tissues like Wharton-Jelly and the human term placenta, UCB advantages, and disadvantages in HSC transplantation, the number of clinical trials applying UCB donation samples, and promising possibilities for regenerative medicine. The existence of different types of UCB-Bs, and the development of new specific cellular manipulation techniques. The UCB-B regulatory framework and some ethical issues were also included in the review. <i>Conclusions:</i> After 30 years of UCB-B, more than 100 public UCB-Bs worldwide, with millions of UCB units donated altruistically, more than 800,000 clinical-grade products are now available: a great source of transplantable SC and other cellular material for the development of new therapies. Even though the UCB as a source of HSC for transplantation has been recently statistical ranked in third place, after HSC apheresis collected and bone marrow aspirates, until now, more than 50,000 UCB HSC allogeneic transplants (HSC-Allo-T) have been performed worldwide in both children and adults to treat many different diseases, including hematologic, metabolic, immunologic, neoplastic, and neurologic disorders. A significant effort has been focused on enhancing engraftment to reduce the risk of infection and cost. <i>Recommendations:</i> To that end, we must understand in detail the molecular mechanisms controlling SC self-renewal that may lead to the development of ex-vivo systems for HSCs expansion, characterize the mechanisms regulating the homing of HSC and determine the relative place of UCB HSC-Allo-T, as compared to other sources.

Bioluminescence Imaging and ICP-MS Associated with SPION as a Tool for Hematopoietic Stem and Progenitor Cells Homing and Engraftment Evaluation.

Bone marrow transplantation is a treatment for a variety of hematological and non-hematological diseases. For the transplant success, it is mandatory to have a thriving engraftment of transplanted cells, which directly depends on their homing. The present study proposes an alternative method to evaluate the homing and engraftment of hematopoietic stem cells using bioluminescence imaging and inductively coupled plasma mass spectrometry (ICP-MS) associated with superparamagnetic iron oxide nanoparticles. We have identified an enriched population of hematopoietic stem cells in the bone marrow following the administration of Fluorouracil (5-FU). Lately, the cell labeling with nanoparticles displayed the greatest internalization status when treated with 30 µg Fe/mL. The quantification by ICP-MS evaluate the stem cells homing by identifying 3.95 ± 0.37 µg Fe/mL in the control and 6.61 ± 0.84 µg Fe/mL in the bone marrow of transplanted animals. In addition, 2.14 ± 0.66 mg Fe/g in the spleen of the control group and 2.17 ± 0.59 mg Fe/g in the spleen of the experimental group was also measured. Moreover, the bioluminescence imaging provided the follow up on the hematopoietic stem cells behavior by monitoring their distribution by the bioluminescence signal. Lastly, the blood count enabled the monitoring of animal hematopoietic reconstitution and ensured the transplantation effectiveness.

Ex Vivo Expansion and Homing of Human Cord Blood Hematopoietic Stem Cells.

Cord blood (CB) has been proven to be an alternative source of haematopoietic stem cells (HSCs) for clinical transplantation and has multiple advantages, including but not limited to greater HLA compatibility, lower incidence of graft-versus-host disease (GvHD), higher survival rates and lower relapse rates among patients with minimal residual disease. However, the limited number of HSCs in a single CB unit limits the wider use of CB in clinical treatment. Many efforts have been made to enhance the efficacy of CB HSC transplantation, particularly by ex vivo expansion or enhancing the homing efficiency of HSCs. In this chapter, we will document the major advances regarding human HSC ex vivo expansion and homing and will also discuss the possibility of clinical translation of such laboratory work.

Stemness‐related genes revealed by single‐cell profiling of naïve and stimulated human CD34+ cells from CB and mPB

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. Here, we applied single-cell RNA-seq (scRNA-seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). We collected over 16 000 high-quality single-cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with cell source (CS-SRGs) and culture time (CT-SRGs), respectively. Interestingly, CS-SRGs and CT-SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT-SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells.

Transient regulation of RNA methylation in human hematopoietic stem cells promotes their homing and engraftment

Enhancing the efficiency of hematopoietic stem cell (HSC) homing and engraftment is critical for cord blood (CB) hematopoietic cell transplantation (HCT). Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of mRNAs that are critical for stem cell function by influencing their stability. Here, we demonstrate that inhibition of RNA decay by regulation of RNA methylation, enhances the expression of the homing receptor chemokine C-X-C receptor-4 (CXCR4) in HSCs. We show that YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), a m6A reader and FTO α-ketoglutarate dependent dioxygenase (FTO), a m6A eraser play an opposite role in this process. Through screening, we identified several FDA-approved compounds that regulate the expression of YTHDF2 and FTO in CB CD34+ cells. We show that transient downregulation of YTHDF2 or activation of FTO by using these compounds inhibits CXCR4 decay in CB HSCs and promotes their homing and engraftment. Our results demonstrate a novel regulation strategy to enhance the function of CB HSCs and provide a translational approach to enhance the clinical efficacy of HCT.

Complement Factor I (CFI) Deficiency Is Linked to Alterations in Humoral Immunity and Accelerated Development of JAK2V617F-Dependent Phenotype

Background: Inflammation is an important phenomenon in hematologic malignancies, though its drivers are not well defined. Within the hematopoietic system, the complement cascade plays a complex role in regulating hematopoietic stem and progenitor cell (hSPC) homing and exit to systemic circulation (Lenkiewicz et al, Frontiers in Immunology 2019). The role the complement system plays in both normal and malignant hSPC egress from the bone marrow has been recognized, though its role in pathogenesis of hematologic malignancies is not well established. Complement cascade contributions to inflammation-mediated pathologies expanded the traditional view of the cascade from an acute process at the site of injury or pathogen invasion, to a mediator of chronic inflammation (Markiewskiet al, American Journal of Pathology 2007). We previously uncovered a rare single nucleotide polymorphism in an inhibitor of the complement cascade Complement Factor I - CFI (CFIG119R). This loss-of-function mutation was detected using whole genome sequencing in 20% of patients with primary myelofibrosis (PMF). In a separate PMF patient cohort, we observed an increase in global complement activity through elevated CH50 levels and decrease in C3 levels (Sadler et al, Blood 2021). There remains a gap in the understanding of how the complement cascade in general and CFI specifically contribute to development and progression of hematologic malignancies. Therefore, we established a murine model of CFI deficiency to determine mechanistic details of complement cascade contribution to both systemic and local inflammation and development of JAK2V617F-dependent pathologies. Methods: To determine the effect of CFI deficiency on homeostasis of hematopoietic system a CFI KO mouse model was generated by floxing exon 2 of the Cfi gene and removing it using CRISPR/Cas9-driven cre recombination. Inactivation of Cfi was confirmed by genotyping. Frequencies of granulocytes, B cells, plasmacytes, T cells, macrophages/monocytes, platelets, erythroid cells were assessed by FACS within the fraction of bone marrow mononuclear cells in 4-week-old CFIWT, HET (n=8) and KO (n=8) mice using established profiling protocols (Chorzalska et al, Blood 2018). Complete blood counts (CBCs) were performed on peripheral blood of 4-week-old Cfi WT (n=20), HET (n=26) and KO (n=10) mice. To determine the effect of CFI deficiency on JAK2V617F-driven phenotypes we performed colony forming assay using CFI WT (n=4, 2F, 2M)) or HET (n=4, 2F, 2M) bone marrows that were transduced with JAK2 V617F encoding retrovirus. Results: CFI KO and HET animals were viable and fertile. CBC analyses of CFI KO, HET vs. WT 4-week-old mice indicated no significant changes with an exception of a significant increase in red blood cell width distribution (RDW) in KO vs. WT and KO vs. HET peripheral blood samples suggestive of a mild anemia. Trends towards granulocytosis and monocytosis as well as leukopenia, erythropenia and decrease in hemoglobin levels were noted. Changes within the red blood cell compartment were confirmed by FSCS. In addition, flow cytometry data showed a significant decrease in CD19+ B-cells in CFI HET vs. WT animals, suggestive of dysregulation of B-cell maturation. Finally, colony forming unit assays showed 59% increase in number of colonies formed by CFI HET/JAK2 V617F+ vs. CFI WT/JAK2 V617F+ bone marrow cells indicative that loss of one copy of Cfi is sufficient to exacerbate JAK2V617F-dependent cellular phenotype. Conclusions: Early characterization of our newly generated CFI KO mouse model shows a phenotype indicative of CFI-loss induced changes within the homeostasis of hematopoietic system affecting red blood cell and B-cell compartments. Our data also indicate that loss of one copy of Cfi may accelerate cell growth and proliferation characteristics of JAK2 V617F-dependent myeloproliferative neoplasms. Detailed analysis of the CFI KO model may uncover novel therapeutically targetable mechanisms that link the pro-inflammatory and myeloproliferative phenotypes of some stem cell disorders, including JAK2 V617F-dependent MPNs.

Case Report: Unique patterns of lymphocyte recovery post-hematopoietic stem cell transplant associated with hyperbaric oxygen therapy: A case series

The utilization of hematopoietic stem cell transplantation (HSCT) has been rapidly growing due to multiple factors, including better availability of donors and improved supportive care. Hyperbaric oxygen has been associated with the improvement of hematopoietic stem cell (HSC) homing at the time of transplant through lowering erythropoietin levels in preclinical studies. We studied the role of hyperbaric oxygen (HBO) in the enhancement of engraftment of HSC when utilized prior to umbilical cord HSCT and autologous HSCT in two pilot clinical trials with excellent safety profiles. In these two pilot studies, we observed an uncommon phenomenon post-transplant, particularly a significant peripheral blood lymphocytosis and lymphocyte infiltration of different tissues in 3/34 of HBO-treated patients. This peripheral blood lymphocyte expansion was associated with various clinical manifestations that can be confused with infections, inflammatory conditions, or disease relapse. We hypothesize that this observation is related to different immune reconstitution dynamics related to the use of HBO. While the incidence is ~9%, this may have implications as HBO is being investigated in larger clinical trials. This case series highlights the clinical presentation, course, outcome, and potential implications of this significant rise in lymphocytes when utilizing HBO before HSCT.

P1349: EFFECT OF TRANSPLANTED CD184-POSITIVE CELLS (WITH EXPRESSION CXCR4) FOR A LONG SURVIVAL AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA – ONE CENTER PROSPECTIVE STUDY.

Background: Autologous hematopoietic stem cell transplantation (AHSCT) remains the treatment of choice in multiple myeloma (MM) patients (pts). The role of chemokine CXCR4 knows also as CXCR4 in hematopoietic stem cell homing engraftment, and in hematopoietic recovery after AHSCT is still not fully understood as well. Aims: In this prospective study we would like to investigate the influence CD184+ cells for overall survival (OS) and progression free survival (PFS) after AHSCT in MM pts. Methods: Forty eight pts with MM with median age 56 (range 21-76) were undergoing AHSCT in our center in years 2011-2013. Conditioning regimen was Melphalan. Number of all CD184+ cells were measured in harvested material. Results: All pts successfully engrafted. The median follow-up was over 8 years. Currently 21 pts (44%) are alive, including 8 pts (17%) in disease’s remission, and 13 are treated for MM progression. Twenty-seven pts died including 19 pts due to disease progression. The median OS was 7.8 years. In the study group a statistically significant correlation was found between OS, PFS and the number of CD184+ cells. In the context of OS and PFS, number of CD184+ cells was found to have a negative impact (correlation coefficient R = -0.32, p=0.0261, and correlation coefficient R = -0.41, p=0.0039 for OS and PFS retrospectively). As OS and PFS increased, the number of CD184+ cells decreased. Summary/Conclusion: The number of CD184+ cells could have a negative effect on both OS and PFS in multiple myeloma patients.

Role of β3-Adrenergic Receptor in Bone Marrow Transplant as Therapeutical Support in Cancer.

β3-adrenergic receptor (β3-AR) is the last β-adrenoceptor subtype identified. β3-AR is widely expressed and regulates numerous physiological processes, and it is also a potential target for the treatment of many diseases, including cancers. For some types of cancers, bone marrow transplant (BMT) represents a valid therapeutic support, especially in the case of the necessity of high-dose chemotherapy and radiotherapy. For a successful BMT, it is necessary that a donor’s hematopoietic stem cells (HSCs) correctly reach the staminal niche in the recipient’s bone marrow (BM) and contribute to restore normal hematopoiesis in order to rapidly repopulate BM and provide all the healthy blood cells of which the patient needs. Generally, it takes a long time. Control and accelerate homing and engraftment of HSCs could represent a helpful approach to avoid the complications and undesirable effects of BMT. The evidence that the β-adrenergic system has a role in the BM can be found in different studies, and this leads us to hypothesize that studying this field could be interesting to meliorate the most critical aspects of a BMT. Here, we collected the data present in literature about the role of β3-AR in the BM with the purpose of discovering a possible utility of β3-AR modulation in regulating HSC trafficking and hematopoiesis.

Myeloablative Conditioning for Transplantation Induces State of Sterile Inflammation in the Bone Marrow: Implications for Optimizing Homing and Engraftment of Hematopoietic Stem Cells.

Significance: The success rate of hematopoietic stem cell transplantation depends mainly on the number of transplanted hematopoietic stem/progenitor cells (HSPCs) followed by the speed of their engraftment in the myeloablated transplant recipient. Therefore, clinical outcomes will significantly benefit from accelerating the homing and engraftment of these cells. This is, in particular, important when the number of cells available for the transplantation of HSPCs is limited. Recent Advances: We postulated that myeloablative conditioning for hematopoietic transplantation by radio- or chemotherapy induces a state of sterile inflammation in transplant recipient peripheral blood (PB) and bone marrow (BM). This state is mediated by activation of the BM stromal and innate immunity cells that survive myeloablative conditioning and respond to danger-associated molecular patterns released from the cells damaged by myeloablative conditioning. As a result of this, several factors are released that promote proper navigation of HSPCs infused into PB of transplant recipient and prime recipient BM to receive transplanted cells. Critical Issues: We will present data that cellular innate immunity arm and soluble arm comprised complement cascade proteins, promoting the induction of the BM sterile inflammation state that facilitates the navigation, homing, and engraftment of HSPCs. Future Directions: Deciphering these mechanisms would allow us to better understand the mechanisms that govern hematopoietic recovery after transplantation and, in parallel, provide important information on how to optimize this process in the clinic by employing small molecular modifiers of innate immunity and purinergic signaling. Antioxid. Redox Signal. 37, 1254-1265.

Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment.

Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.

Alterations in Hematopoietic and Mesenchymal Stromal Cell Components of the Osteopetrotic Bone Marrow Niche.

Osteopetrosis is a rare inherited disease characterized by impaired osteoclast activity causing defective bone resorption and bone marrow aplasia. It is fatal in early childhood unless hematopoietic stem cell transplantation is performed. But, the transplant course is complicated with engraftment failure. Recently, osteoclasts have been described as the potential regulators of hematopoietic stem cell (HSC) niche. Here we investigated the alterations in the HSC and mesenchymal stromal cell (MSC) components of osteopetrotic niche and their interactions to mimic the stem cell dynamics/trafficking in the BM niche after HSC transplantation. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells of patients with osteopetrosis carrying TCIRG1 mutation. iPSC lines were differentiated into hematopoietic and myeloid progenitors, then into osteoclasts using a step-wise protocol. We first demonstrated a shift toward monocyte-macrophages lineage regarding hematopoietic differentiation potential of osteopetrotic iPSC-derived hematopoietic progenitors (HPCs) and phenotypically normal and functionally defective osteoclast formation. The expression of the genes involved in HSC homing and maintenance (Sdf-1, Jagged-1, Kit-L, and Opn) in osteopetrotic MSCs recovered significantly after coculture with healthy HPCs. Similarly, the restoration of phenotype, impaired differentiation, and migratory potential of osteopetrotic iHPCs were observed upon interaction with healthy MSCs. Our results establish significant alterations in both MSC and HPC compartments of the osteopetrotic niche, and support the impact of functionally impaired osteoclasts in defective niche formation.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN‐γ Signaling (Adv. Sci. 5/2022)

Hematopoietic Stem and Progenitor Cells In article number 2103838, Xiao-Ming Yang, Chang-Yan Li and co-workers identify a critical regulator-HEMGN, that is required for hematopoietic stem and progenitor cells (HSPCs) homing, survival, and reconstitution of blood cells during bone marrow transplantation (BMT). HEMGN acts as a protector which limits interferon-γ (IFN-γ) signaling by suppressing nuclear Stat1 tyrosine phosphorylation and assists HSPCs to combat the transplantation stress. Targeted Hemgn may be used to improve conditioning regimens and engraftment during bone marrow transplantation.

Microtubule Polymerization Inhibition Enhances Human Hematopoietic Stem Cell Homing and Engraftment

Hematopoietic stem cells (HSCs) serve as the origin of the hematopoietic system, with the ability to differentiate into all blood cell lineages and self-renewal to sustain the hematopoiesis throughout life. Hematopoietic stem cell transplantation (HSCT) currently represents the most effective therapeutic strategies to treat hematological and non-hematological diseases. However, limited numbers of HSCs or poor homing capabilities into the bone marrow are still major hurdles for successful HSCT. Moreover, graft failure and delayed reconstitution due to inefficient engraftment, remains an important complication because of the high morbidity and mortality. Although ex vivo expansion of HSCs has been well studied for decades, which displays huge potentials for clinical application, exploration of novel targets to improve HSC homing and engraftment will provide new insights to enhance HSCT efficacy.To explore the chemical compounds enhancing the capabilities of homing and engraftment, we used CXCR4 (CD184) as the readout bio-marker, which is considered as the most essential chemokine receptor of HSPCs interacting with CXCL12 (SDF1) secreted in BM niche to support HSPCs homing, migration, proliferation and survival. We first performed chemical screening of 139 small molecules that can increase CD184 expression on cord blood (CB) CD34 + hematopoietic stem and progenitor cells (HSPCs). We concluded that treatment of CB CD34 + HSPCs for 16 hours with Lexibulin (Lex) or Vinblastine Sulfate (VS), both of which were microtubule polymerization (MP) inhibitors, could significantly promote the CD184 expression.Next, we optimized the MP inhibitors treatment conditions including dosage, treatment duration and culture time prior to treatment. The results proved that treatment with Lex or VS for 16 hours at 1μM was the optimal conditions to significantly enhance the CD184 expression of CD34 + HPSCs and LT-HSCs (CD34 +CD90 +CD45RA -), while maintaining robust cell survival, when compared with the DMSO control group. Moreover, we found that only when HSPCs were under culture within two days prior to small molecules treatment, CD184 expression was significantly increased by MP inhibitors while maintaining high viability, compared with DMSO control group.In order to assess the in vivo repopulating potential of the CB CD34 + HSPCs post treatment with MP inhibitors, we transplanted CB-HSPCs 16 hours post-treatment with Lex and VS respectively into irradiated nonobese diabetic (NOD)/Prkdc scid/IL-2Rγ null (NPG) mice. All transplanted mice of MP inhibitors-treated groups presented efficient engraftment, in multiple immune organs at 4-16 weeks post-transplantation, suggesting greater engraftment potential than the mock group, as measured by human CD45 of total CD45. Furthermore, hematopoietic reconstitution analysis indicated that the MP inhibitors -treated cells maintained different lineage distribution in peripheral blood (PB), bone marrow (BM) and spleen. Moreover, the equivalent phenotypes of pre- and post-treatment reveal the better reconstitution by MP inhibitors was independent of HSC-enrichment, Thus, short-term MP inhibitors treatment of CB CD34 + HSPCs enhances their homing and long-term engraftment.In conclusion, we demonstrated that short-term microtubule polymerization inhibition on human CB CD34 + HSPCs could not only enhance CD184 cell surface expression but also the capabilities of in vivo human HSCs homing and reconstitution via screening chemical compounds to increase CD184 expression and the following function evaluation study. Vinblastine Sulfate and Lexbulin were applied or registered as anti-cancer drugs for clinical use. Our study also indicates that MP inhibitors pretreatment of cells possesses significant translational implications, designating MP inhibitors as promising drug candidates to facilitate clinical HSCT. [Display omitted] DisclosuresFang: EdiGene, Inc.: Current Employment.