Hematopoietic Stem Cell Content Research Articles

Intracellular Iron Overload Induces Metabolic Switching in Active Hematopoietic Stem Cells in Beta-Thalassemia

Beta-thalassemia (BThal) is a complex monogenic disorder, causing severe anemia, ineffective erythropoiesis and multi-organ secondary defects. Iron overload (IO), associated with ineffective erythropoiesis and therapeutic blood transfusions, is one of the main complications of the disease, leading to morbidities in various organs. Despite the improvement in chelation therapies, IO toxicity is still a relevant issue. Correction of BThal is achieved by transplantation of hematopoietic stem cells (HSC) from healthy donors or autologous HSC from patients upon gene therapy. The quality and the engraftment of HSC depend on the bone marrow (BM) microenvironment, thus niche-HSC crosstalk plays a crucial role for transplantation outcome. We provided the first demonstration of impaired function of HSC caused by an altered BM niche in BThal (Aprile et al., Blood 2020). HSC from Hbbth3/+ (th3) BThal mice showed more active cycling profile and response to stress. We reported that IO reduces the hematopoietic supportive capacity of BM mesenchymal stromal cells from patients and recent evidence highlighted that both IO and iron deficiency are sources of cellular stress detrimental to HSC maintenance. Thus, we hypothesized that accumulation of iron might directly affect HSC function in BThal, as a model of chronic IO. Transcriptome analysis of sorted HSC from th3 mice showed a positive enrichment of genes involved in iron homeostasis, such as TfR1, Steap3, Fth1 and Ftl1, suggesting iron uptake and storage. Consistently, BThal HSC displayed higher levels of intracellular free reactive iron, as compared to wild-type (wt) controls (1.7±0.2 calcein MFI fold to wt, p<0.0001). We found that iron specifically accumulates in the mitochondria causing mitochondrial defects, as reduced size and lower membrane potential (0.4±0.05 TMRE/MTG fold to wt, p<0.001). These data, along with the downregulation of mitochondrial biogenesis and mitophagy genes, indicate an accumulation of damaged mitochondria in BThal HSC. In vivo administration of iron dextran to wt mice generated intracellular IO in HSC and was sufficient to decrease the mitochondrial membrane potential at levels comparable to those of th3 HSC, thus suggesting a direct effect of iron on mitochondrial activity. Since mitochondria are the powerhouse of the cell, we wondered if the mitochondrial dysfunction of BThal HSC could affect their production of metabolic energy. Metabolism indeed control HSC function: active HSC are more cycling and, because of their higher energy request, change the metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, we found that ATP levels in th3 HSC are lower compared to controls (wt 9.8±2.6 vs. th3 3.7±0.3 ATP nM, p<0.01). In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP content of BThal HSC since they switch to glycolysis for their energy demand. Consistently, th3 HSC showed positive enrichment of glycolytic genes and higher glucose uptake (wt 1639±163 vs. th3 2401±85 MFI 2NBDG, p<0.05). To unravel the basis of mitochondrial damage caused by IO, we investigated the role of reactive oxygen species (ROS). Iron is one of the sources of ROS and consistently, th3 HSC showed increased activation of antioxidant genes and higher levels of mitochondrial ROS (wt 2507±756 vs. th3 5205±459 MFI MitoSOX, p<0.01). In vivo reduction of mitochondrial ROS by MitoQ rescued mitochondrial activity (th3 0.3±0.07 vs. th3+MitoQ 0.9±0.2 TMRE/MTG fold to wt, p<0.05), thus demonstrating that mitochondrial dysfunction is reversible in BThal. Ongoing experiments will assess the metabolite profile, the restoration of HSC metabolism and in vivo function upon MitoQ and will also test HSC from BThal patients for mitochondrial defects. Overall, our study revealed that the multisystemic complications of IO in BThal also affect HSC. Iron has a direct impact on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Despite the active cycling state, BThal HSC switch their metabolism to glycolysis because of the mitochondrial defect. Targeting mitochondrial iron or ROS might provide new tools to preserve HSC function in the setting of autologous transplantation upon gene therapy/editing for BThal. Moreover, this research will add novel insight about the regulation of HSC biology linking iron to mitochondrial metabolism.

Pluripotin stimulates the growth of hematopoietic stem cells while suppressing the expansion of bone marrow mesenchymal stem cells and fibroblasts

Hematopoietic stem cells (HSCs) are essential in the production and maintenance of red blood and immune cells. Small molecules that target HSC modulators may aid in the proliferation and expansion of HSCs. To that end, we investigated the effect of two small molecules on HSC expansion: pluripotin (an ERK1 and RasGAP inhibitor) and CHIR-99021 (a GSK-3 inhibitor). After 7 days of treatment, both Pluripotin and CHIR-99021 resulted in a 3-fold increase in the murine pool of HSCs in a dose-dependent manner. Furthermore, we looked into the effect of Pluripotin on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. Pluripotin treatment, in particular, increased human CD34+ and ALDHbr HSC content up to threefold when compared to the control. Furthermore, Pluripotin treatment increased the number of human CD133+ HSC cells by a factor of five. Intriguingly, Pluripotin treatment reduces bone marrow-derived mesenchymal stem cell (MSC) proliferation and fibroblast growth while having no effect on adipose-derived MSCs. CHIR-99021 treatment had no effect on MSC or fibroblast proliferation. In conclusion, pluripotin-induced stem cell expansion is unique to HSCs and can be used to expand HSCs while suppressing unwanted fibroblast or MSC growth in primary ex vivo cultures.

Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells.

Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB‐HSC is represented by the use of bone marrow mesenchymal stromal cells (BM‐MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM‐MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM‐MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB‐HSC was evaluated in comparison to the control condition represented by the CB‐CD34+ cells without a feeder layer. The coculture was performed at two different CD34+:MSC ratios (1:2 and 1:8) in both cell‐to‐cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM‐MSC to support: (a) the ex vivo expansion of CB‐HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long‐term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation.

Enhanced Free Fatty Acid Uptake Via CD36 Promotes a Metabolic Switch to B-Oxidation within Hematopoietic Stem Cells in Response to Acute Infection

Acute infection is known to promote rapid differentiation of hematopoietic stem cells (HSC) and expansion of leukocytes. Metabolic changes in the HSC underpin the mammalian response to pathogenic stimuli however, knowledge of how this occurs is not fully understood. Here we investigate the immunometabolic changes which facilitate HSC expansion in response to acute infection. We created a novel in vivo transplant model to understand if the immune response to infection involves the acquisition of free fatty acids (FFA) by HSCs in the bone marrow (BM). We transduced CD45.1 lineage negative, c-kit positive cells with firefly luciferase and transplanted into CD45.2 animals. Post engraftment animals were treated with lipopolysaccharide (LPS). Live animal imaging confirmed activation of luciferase in the BM, demonstrating in vivo, uptake of long chain FFA by haematopoietic cells in response to LPS. To confirm the specific haematopoietic stem and/or progenitor cells (HSPC) with increased lipids during infection, mice were treated with S.typhimurium for 72h then sacrificed Analysis of LSK, MPP and HSC populations (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) showed increased intracellular BODIPY 493/503 neutral lipid dye and uptake of Bodipy FL-C12 (FFA linked to bodipy) at 72 h compared to control non-infected animals. Together, these experiments show that HSC, multipotent progenitor (MPP) and LSK (LN-, CD117+, Sca1+) cells all acquire FFA in response to bacterial infection. Seahorse mitochondrial stress test confirmed increased oxygen consumption levels in HSC from LPS (16 hours) and S.typhimurium (72 hours) treated C57BL/6. LSK metabolic activity for different substrates was assessed LSKs from LPS treated animals had an increased dependency on β-oxidation when compared to control cells. Moreover, the β-oxidation inhibitor etomoxir blocks LSK basal metabolism and reduced HSC expansion as measured by Ki-67 staining in LPS and S.typhimurium treated animals. To understand the importance of β-oxidation in HSC expansion in response to infection we first measured CPT1A expression in HSC. RNA analysis confirmed CPT1A was increased in sorted HSCs in response to LPS. Next we transplanted WTor CPT1A KD cells into WT animals, and treated with LPS. HSC expansion as measured by Ki-67 staining was inhibited in the CPT1A KD cells in response to LPS, thus confirming the importance of β-oxidation based metabolism in HSC expansion. To determine how FFA are transported into the HSC we analysed the expression of known genes to be involved in lipid uptake. HSC sorted from LPS and S.typhimurium treated animals had increased CD36 mRNA expression compared to control. C57BL/6 mice were pre-treated with the CD36 inhibitor sulfosuccinimidyl oleate (SSO) before injection with LPS. HSCs had had less lipids, lower maximal respiration and reduced cycling when compared to animals treated with LPS alone. In a similar way CD36 KO animals were treated with LPS for 16 h, no increased FFA uptake or increased lipid content or HSC cycling was observed. Seahorse mitochondrial stress test confirmed CD36-/- LSK have increased basal ECAR but no change in basal OCR in response to S.typhimurium infection. Moreover, CD36-/- LSK have reduced dependency on β-oxidation. To determine if the uptake of FFA response is specific to HSC we transplanted CD36+/+ (CD45.1) LK into CD36-/- (CD45.2) animals. We found that CD36 expression was elevated in CD36+/+ HSC transplanted into CD36-/- treated with LPS, normal uptake of FFA, an increase in lipid content and cycling of HSCs. In addition, transplanted WT CD36+/+ (CD45.1) LSK into CD36-/- (CD45.2) animals reversed the metabolic phenotype observed in CD36-/- in response to LPS. We transplanted WT(CD36+/+) or CD36KO (WT(CD36-/-)) LSK into WT animals infected with S. typhimurium for 4 days. WT(CD36-/-) transplanted animals showed enhanced mortality, increased weight loss and increased liver injury. We also observed less HSC cycling and a reduced lipid content in HSC from WT(CD36-/-) compared to WT(CD36+/+). These findings expand the mechanistic understanding of the interplay between HSC and the bone marrow microenvironment, and in doing so explains how immunometabolic reprogramming of HSCs during infection supports the metabolic demands of HSCs in response to a pathogenic challenge without which results in increased susceptibility to infection. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:AbbVie: Research Funding; Janssen: Research Funding.

Development of Small Molecule MEIS Inhibitors that modulate HSC activity

Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1α, Hif-2α and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca2+ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K+ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.

Mobilization regimen, including Plerixafor, does not impact CD34 recovery after automated post-thaw processing

Background & Aim Autologous Hematopoietic stem cell (HSC) transplantation is still widely used to overcome hematologic toxicity after high dose chemotherapy for patients with hematological malignancies such as multiple myeloma or lymphoma among others. In most cases, HSC are collected by apheresis after an appropriate mobilization regimen. At our institution, cryopreserved autologous grafts are thawed and washed before infusion. All these processing steps can be performed manually or via automated processes or devices. We aimed to evaluate the impact of mobilization regimen on rh-G-CSF or Plerixafor graft quality when using standardized, automated processes. Methods, Results & Conclusion Methods Between 2016 and 2019, 535 patients received an autologous transplant at our institution. From this cohort, we extracted 260 patients (n=170 myeloma and n=90 myeloma) for whom processing of the apheresis product was comparable, i.e. cryopreservation the same day of the HSC procurement in 2 bags, standardized thawing using Smartmax followed by automated washing using the Sepax-2. Among this cohort, 90 patients (n=75 myeloma and n=15 lymphoma) received Plerixafor + rh-G-CSF as a mobilization regimen, and 170 patients (n=95 myeloma and n=75 lymphoma) received a standard regimen (either rh-G-CSF alone or rh-G-CSF in addition to chemotherapy for lymphoma). Measurable outputs were CD34+ cells/ kg before and after thawing, CD34+ cells recovery and viability, CD45+ cells/kg, neutrophils and myelemia before cryopreservation and CD45+ cells viability after thawing. Results Apheresis products collected from patients mobilized with Plerixafor versus rh-GCSF showed lower HSC content (3,2 × 106/kg versus 5 × 106/kg respectively) and lower myelemia (7 × 106/kg versus 12,9 × 106/kg respectively, p=0.005). Total CD45+ and neutrophils contents were similar (median 50 × 109 and 3 × 109 respectively, p = NS). CD45+ and CD34+ viabilities were comparable between the two regimen (around 83% and 85% respectively p = NS). For both groups, viable CD34+ cells recovery was similar (median 79% for the plerixafor group versus 76% for the rh-G-CSG group p = NS). Conclusions Our data show that CD34+ cells recovery is not affected by the nature of mobilization agents when using standardized automated processing post-cryopreservation.

Evaluation of the Sysmex XN automated hematopoietic progenitor cell enumeration for timing of peripheral blood stem cell harvest

BackgroundEnumeration of stem cells is essential in the management of peripheral blood stem cell (PBSC) harvest. An alternative to the gold standard flow cytometric CD34+ stem cell count is the fully automated hematopoietic stem cell (HPC) count on the Sysmex XN hematology analyzer. Materials and methodsEighty-nine patients and healthy stem cell donors who underwent PBSC harvest were included in the study. Stem cells were enumerated in pre-harvest peripheral blood and the apheresis yield by both flow cytometric CD34+ stem cell enumeration and by the Sysmex XN HPC count. ResultsThe Sysmex HPC concentration overestimated the CD34+ stem cell concentration by a ratio of 1.3 in average. The agreement between the two methods was poor at concentration <40 stem cells/μL (Bias: 45 %, 95 % limits of agreement: -71 - 160 %). CD34+ stem cell concentration and HPC concentration correlated well in pre-harvest peripheral blood (R=0.73, slope=0.96). We established a positive cut off >43.5 HPC/μL, where PBSC harvest can be initiated. And a negative cut off <16.5 HPC/μL, where harvest should be postponed or other mobilizing regimens or bone marrow harvest should be considered. 33 % of measurements were in between the negative and positive cut-off and would require a supplementary CD34+ cell count. ConclusionAlthough Sysmex HPC count correlates well with CD34+ cell count in peripheral blood, the agreement between the two methods is poor, especially at low concentrations, namely in the clinical decision range. Sysmex HPC count as a surrogate for CD34+ cell count should, therefore, be used with caution.

Cryopreservation of placenta tissue allows isolating viable mesenchymal and hematopoietic stem cells

Background & Aim Cryopreserved placenta tissue could be prospective source of mesenchymal and hematopoietic stem cells for cell therapy and regenerative medicine. The aim of our study was to determine methods of placental cryopreservation with high stem cells recovery. Methods, Results & Conclusion Chorions (n=4) were isolated from full-term human placentas of healthy donors. One part of tissue from every specimen was seeded as a native control and the other part was frozen. Nine variants of cryopreservation medium composition were selected by combination of different concentration of dimethyl sulfoxide (5%, 7.5%, 10%) and sucrose (0.1 M, 0.2 M) as penetrating and non-penetrating cryoprotectants. The samples were frozen in cryogenic vials in a controlled rate freezer (IceCube, Austria). Placenta-derived multipotent cells (PDMCs) were subcultured from native and thawed chorion explants. Colony forming units (CFU) assay was carried out on passages 0, 2, 6 for PDMCs isolated from native and thawed tissue. Hematopoietic stem cells (HSCs) of full-term human placental tissue were obtained by enzymatic method with following depletion of dead cells. Immunophenotyping was performed on BD FACSAria (USA). MethoCult (StemCell Technologies, Canada) was used for differentiation assays. XY karyotype of PDMCs indicates their foetal origin. The results of flow cytometry analysis revealed CD90+ CD105+ CD73+ CD34− CD45− PDMCs phenotype. PDMCs were able to differentiate into osteogenic and adipogenic lineages. CFU assay showed that number of native tissue-derived colonies on passage 0 was significantly higher in comparison with thawed tissue for the nine variants of cryoprotectants. However, number of CFU for fresh-isolated cells on passages 2 and 6 was not significantly differ in comparison with PDMCs from thawed tissue. Flow cytometry analysis showed that the HSCs content among CD45+ cells of the total light-density cell fraction from the cryopreserved and native placental tissue did not significantly differ. CFU study demonstrated the presence of myeloid progenitors, quantitative analysis revealed difference in efficiency of HSCs preservation between certain variants of cryoprotectants. The best variant reached up to 50% of CFU recovery compared to native sample. Controlled cryopreservation of placental tissue under GMP requirements allows to obtain proliferative active stem cells with suitable for clinical issues characteristics.

Synthesis and evaluation of pyrimidoindole analogs in umbilical cord blood ex vivo expansion

The scarcity of hematopoietic stem cells (HSCs) significantly hindered their clinical potentials. Umbilical cord blood (UCB) has become the leading source of HSCs for both research and clinical applications. But the low content of HSCs in a single UCB unit limited its use only to pediatric patients. Various cytokines and small molecules have demonstrated strong abilities in promoting HSC ex vivo expansion, of which UM171 is the newest and by far the most potent HSC ex vivo expansion agent. In this study, we synthesized 37 pyrimidoindole analogs and identified 6 compounds to be potent in promoting HSC ex vivo expansion. In particular, analog 11 was found to be the most effective in stimulating ex vivo expansion of UCB CD34+ cells and CD34+CD38− cells. Initial data indicated that compound 11 promoted the absolute number of long term HSCs and inhibited their differentiation. UCB HSCs expanded with 11 retained adequate multi-lineage differentiation capacity. In addition, compound 11 is not cytotoxic at its test concentrations, suggesting that it merits further investigation for potential clinical applications.

CD117-Amanitin Antibody Drug Conjugates Effectively Deplete Human and Non-Human Primate HSCs: Proof of Concept As a Targeted Strategy for Conditioning Patients for Bone Marrow Transplant

Background: Genotoxic conditioning prior to allogeneic and autologous bone marrow transplantation (BMT) limits the use of these potentially curative treatments due to risks of regimen-related morbidities and mortality, including risks of organ toxicity, infertility, and secondary malignancies. CD117, which is specifically expressed on hematopoietic stem cells (HSCs) and progenitors is rapidly internalized and is an ideal target for an antibody drug conjugate (ADC) based approach to conditioning. We have previously shown that a single dose of an anti-CD117 ADC depleted >95% of bone marrow HSCs in a humanized mouse model and reduced disease burden while extending survival in an AML tumor model (Hartigan et al., Blood 2017 130:1894). The aim of this translational study was to develop a potent anti-CD117 ADC highly effective in eliminating host HSCs with a short half-life and minimal adverse side effects in a non-human primate (NHP) model.Methods: Three different DNA-damaging cytotoxic payloads and amanitin (AM) were site specifically conjugated to an anti-CD117 antibody. The ADCs were titrated and evaluated for in vitro cytotoxicity using human bone marrow CD34+ cells. The ADCs were administered in ascending doses to humanized NSG mice. HSC depletion and immunophenotype of the human cells in the peripheral blood was determined by flow cytometry. For amanitin conjugates, NHP HSC depletion was evaluated in male cynomolgus monkeys in single ascending doses (3/group). HSC content in the bone marrow was monitored by flow cytometry and colony-forming unit (CFU) analysis on day 7 or 14 and 56 post dosing. Hematology and clinical chemistries were evaluated throughout the two-month study.Results: Of the toxins evaluated, only anti-CD117 conjugated with the RNA polymerase II inhibitor amanitin resulted in >90% depletion of human HSCs in humanized NSG mice at 0.3 mg/kg. The AM-conjugates also demonstrated a broad therapeutic window in this model (therapeutic index of >120). As a proof-of-concept for the depletion of HSCs in large animals, a single i.v. dose escalation study was performed with the cross-reactive anti-CD117-AM in NHP. On-target, dose-dependent decreases in phenotypic HSCs and CFUs were observed in the bone marrow at day 7 post anti-CD117-AM dosing with >95% HSC depletion observed with a single dose of 0.3 mg/kg (Fig. 1). In the periphery, a dose-dependent transient decrease in reticulocytes was observed at day 4 with a neutrophil and monocyte nadir at day 18. The depth and duration of the depletion was also dose-dependent. The anti-CD117-AM induced depletion was on target and amanitin dependent as the unconjugated antibody and isotype-AM had no effect. Notably, white blood cell and lymphocyte counts were stable through day 56, demonstrating that this strategy will spare the adaptive immune system. Thrombocytopenia occurred 4-8 days post infusion and was dose-dependent, transient and reversible. This also occurred with the isotype-AM, suggesting the effect was off-target.Because the half-life of the anti-CD117-AM was 5 days, a second dose escalation study with anti-CD117-AM engineered to have a short half-life (~18 h) was performed in NHPs. The short half-life anti-CD117-AM demonstrated similar potency on all cell parameters at 0.3 mg/kg and was well tolerated at the effective dose. As expected, the short half-life anti-CD117-AM was rapidly cleared with a half-life of 15-18 h. In both studies, a transient dose dependent elevation of liver enzymes was observed in groups treated with the highest doses of isotype-AM, anti-CD117-AM, and the short half-life anti-CD117-AM.Conclusions: Anti-CD117-AM exhibited potent elimination of NHP HSCs and progenitors in vivo. The potency of short half-life anti-CD117-AM was comparable, providing a model for target cell depletion and rapid clearance prior to BMT. Both ADCs were well tolerated at the efficacious doses. This strategy preserves the adaptive immune system with delayed onset of neutrophil nadir (18 days), potentially shortening the period of neutropenia. Targeted depletion of hematopoietic cell subtypes with limited off-target effects could provide a significant improvement in standard-of-care approaches to patient preparation prior to HSC transplant. [Display omitted] DisclosuresPearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Kien:Magenta Therapeutics: Employment, Equity Ownership. Dushime:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Kallen:Magenta Therapeutics: Employment, Equity Ownership. Sawant:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient's age and harvesting technique.

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45-/CD73+/CD90+/CD105+ and CD45-/CD271+) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45-/CD271+ cells correlates better with CFU-F numbers than the population of the CD45-/CD73+/CD90+/CD105+ cells. The relationship between the hematopoietic CD45dim/CD34+ cell concentration and mesenchymal CFU-Fs or CD45-/CD271+ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45dim/CD34+, mesenchymal CD45-/CD73+/CD90+/CD105+ and CD45-/CD271+ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells.

Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Stem Cell Yield after Bone Marrow Concentration

Objectives:The number of Mesenchymal Stem/Stromal Cells (MSCs) in the human bone marrow (BM) is small compared to other cell types, estimated at 0.01-0.02%. Bone marrow aspirate concentration (BMAC) may be used to increase numbers of MSCs, but the efficacy of BMAC, as well as the composition of MSC subpopulations after processing, is currently unknown. The purpose of this descriptive laboratory study is to assess the enrichment of MSC subpopulations in bone marrow aspirate by two different BMAC devices versus standard BM aspiration from single donors.Methods:120 mL of BM was aspirated from multiple punctures of the iliac crest in 9 human male donors. Each sample was divided into 3 partitions and processed by 2 different commercially available systems Harvest BMAC or Emcyte PureBMC, and compared to untreated BM aspirate as an internal control. Samples were quantitatively analyzed with multicolor flow cytometry for cellular viability and expression of hematopoietic and MSC subpopulation markers. Colony-forming unit-fibroblasts (CFU-F) assays were performed to estimate the number of functional progenitor cells.Results:Cell viability after processing was over 90% in all groups without significant differences, and hematopoietic cell content was lower in BMAC where, in contrast, non-hematopoietic stem cell subpopulations were significantly enriched. Specifically, BMAC from Harvest BMAC contained more CD45-CD73+CD90+ (11.44 fold vs control, p = 0.01), CD45-CD10+ (3.68 fold vs control, p = 0.01), CD45-CD29+ (1.54 fold vs control, p = 0.03), and CD45-CD119+ (5.52 fold vs control, p = 0.02) cells, whereas Emcyte PureBMC concentrated more CD45-CD73+ cells (13.90 fold vs control, p = 0.04). Both BMAC devices mainly enriched the CD45-CD90+CD271+ MSC subpopulation (Harvest BMAC: 13,011.84 fold vs control, p = 0.001; Emcyte PureBMC: 10,669.11 fold vs control, p = 0.02) compared to other MSC subpopulations. Both BMAC systems significantly increased progenitor colonies (Harvest BMAC: 2692 CFU-F/mL; Emcyte PureBMC: 4336 CFU-F/mL) verses control BM aspiration (183 CFU-F/mL), p<0.05.Conclusion:BMAC devices lead to: 1) enrichment of functional mesenchymal stem cell subpopulations with distinct phenotypes, 2) no increase in hematopoietic stem cell concentration, and 3) no significant loss of cell viability.

Depth-dependent concentrations of hematopoietic stem cells in the adult skeleton: Implications for active marrow dosimetry.

The hematopoietically active (or red) bone marrow is the target tissue assigned in skeletal dosimetry models for assessment of stochastic effects (leukemia induction) as well as tissue reactions (marrow toxicity). Active marrow, however, is in reality a surrogate tissue region for specific cell populations, namely the hematopoietic stem and progenitor cells. Present models of active marrow dosimetry implicitly assume that these cells are uniformly localized throughout the marrow spaces of trabecular spongiosa. Data from Watchman etal. and Bourke etal., however, clearly indicate that there is a substantial spatial concentration gradient of these cells with the highest concentrations localized near the bone trabeculae surfaces. The purpose of the present study was thus to explore the dosimetric implications of these spatial gradients on active marrow dosimetry. Images of several bone sites from a 45-yr female were retagged to group active marrow voxels into 50μm increments of marrow depth, after which electron and alpha-particle depth-dependent specific absorbed fractions were computed for four source tissues - active marrow, inactive marrow, bone trabeculae volumes, and bone trabeculae surfaces. Corresponding depth-dependent S values (dose to a target tissue per decay in a source tissue) were computed and further weighted by the relative target cell concentration. These depth-weighted radionuclide S values were systematically compared to the more traditional volume-averaged radionuclide S values of the MIRD schema for both individual bones of the skeleton and their skeletal-averaged quantities. For both beta-emitters and alpha-emitters localized in the active and inactive marrow, depth-weighted S values were shown to differ from volume-averaged S values by only a few percent, as dose gradients across the marrow tissues are nonexistent. For bone volume and bone surface sources of alpha-emitters and lower energy beta-emitters, when marrow dose gradients are expected, explicit consideration of target cell spatial concentration gradients are shown to significantly impact marrow dosimetry. For medical isotopes currently utilized for treatment of skeletal metastases, namely 153 Sm and 223 Ra, accounting for hematopoietic stem and progenitor cell concentration gradients resulted in maximum percent differences to reference skeletal-averaged S values of ~21% and 55%, respectively.

COMPARATIVE CHARACTERISTICS OF THE BONE MARROW FROM FEMORAL HEAD AND ILIAC BONE IN PATIENS WITH FEMORAL NECK FRACTURES

Increasing number of elderly patients with femoral neck fractures requires a search for an optimal treatment regimen based on a natural sustenance and regulation mechanisms of bone homeostasis.Purpose of the study – to perform a comparative cellularity analysis of bone marrow obtained from femoral neck and iliac bone of elderly patients.Material and methods. The experimental group included 29 patients with closed fractures of femoral neck, injured by falling on the side from a standing position. Bone marrow was obtained from femoral neck and iliac bone during procedure of screw internal fixation or hip replacement. In the control group, the authors used bone marrow harvested from the same anatomical areas of 10 cadaveric donors. Nucleated cells (NCs), hematopoietic stem cells (HSCs) and multipotent mesenchymal stem cells (MSCs) were counted in bone marrow by flow cytometry.Results. Comparative study demonstrated that bone marrow from the hip of patients with fractures and cadaveric donors contained less concentration of NCs and HSCs than bone marrow from the ilium. However, the difference in concentration was significant only in the experimental group. At the same time, in patients with fractures the NCs concentration and absolute content of HSCs in bone marrow obtained from hip was significantly lower than those from cadaveric donors. Meanwhile, samples obtained from the iliac bone in the experimental and control groups had no significant difference. The authors also observed that concentration of HSCs and MSCs in the bone marrow from male hips was significantly lower than in females. As a result, male patients with fractures demonstrated difference in concentrations of HSCs samples from ilium and femoral neck reaching 84.6%, MSCs – 86.6%; females demonstrated differences of 68.9% and 69.2% respectively.Discussion. The data collected allows suggesting that one of the main factor violating micro-architecture of bone and in development of osteoporosis is an alteration in the cellular composition of bone marrow. While MSCs are active in regeneration of bone tissue, increase in their concentration in the fracture zone can stimulate the healing. Thus, to improve treatment outcomes in patients with hip fractures after internal fixation with cannulated screws, it could be beneficial to use grafting of autologous bone marrow obtained from the iliac bone featured by a higher stem cells concentration.

Editorial: "How to Improve Cord Blood Transplantation: By Enhancing Cell Counts or Engraftment?".

Editorial: "How to Improve Cord Blood Transplantation: By Enhancing Cell Counts or Engraftment?".

Connexins: Intercellular Signal Transmitters in Lymphohematopoietic Tissues.

Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.

Ex Vivo Pharmacologic Modulation in a Nutrient-Rich Medium to Accelerate Engraftment of Human Umbilical Cord Blood

Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality.Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing [North 2007&2009; Hoggatt 2009]. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®).A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) [Cutler 2013]. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months.During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change. DisclosuresShoemaker:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.

Plasma CC-chemokine ligand 28 level is correlated with hematopoietic stem cells in human cord blood.

CC-chemokine ligand 28 (CCL28) was previously identified as a novel growth factor in vitro for hematopoietic stem cells (HSCs) from cord blood (CB). However, there is no report on the relationship between CCL28 and HSCs in a human body. To reveal the effect of CCL28 on hematopoietic cells in human CB at birth, we measured CCL28 in frozen CB plasma, which was preserved as a reference sample for cryopreserved CB units for HSC transplantation. We also evaluated the correlation of CCL28 level with CB components. A total of 81 cryopreserved nonconforming CB units for transplantation were selected. The level of CCL28 was 2540 ± 377 pg/mL. The CCL28 levels correlated with the number of CD34+ cells (r = 0.222, p = 0.047) and white blood cells (r = 0.254, p = 0.022) in the CB units. The CCL28 levels also correlated with hemoglobin levels (r = 0.221, p = 0.048) in fresh CB. This finding of positive correlation between CCL28 level and CD34+ cell numbers in vivo, together with the previous report that CCL28 influences the proliferation of hematopoietic cells in CB in vitro, may give a clue for better understanding the variability in HSC content in CB that is cryopreserved for HSC transplantation.