Human Cord Blood-derived Stem Cells Research Articles

Abstract 4493: Protein structure inspired drug discovery

Abstract Introduction: Drug action is mediated by a small molecule therapeutic binding to a specific structural motif on a protein. As structure determines function, it follows that unique structural motifs are enriched for sites of unique function and high therapeutic target value. We hypothesized that protein structure constitutes high value primary information that can drive the discovery of novel therapeutic agents. Method: We developed a unique physics-based protein structure analysis platform scaled to run on a supercomputer, allowing us to consider dynamic protein flexibility and the influence of solvent upon protein structure, and to probe large protein libraries as a primary source of structure information that can support up-front structure-based screens. We developed and then probed a high resolution protein x-ray crystallographic library, created and curated by us. From a 60 million compound library, we conducted virtual screens for docking to identified sites. 8-day colony formation assays were conducted on a panel of 8 human breast, prostate, lung and colon cancer cell lines, as well as 8- and 14-day trilineage hematopoietic colony formation assays on human cord blood stem cells. Findings: From the first 180 deposited protein structures, we screened for unique protein surface structure. Structures were deemed unique if they were not otherwise known to be associated with any particular functional roles and were not the binding site of known therapeutics. Further selection included those that were clefts and possessed physical characteristics compatible with binding small chemicals whose properties have been linked to effective therapeutics. For each of 8 sites on 6 different proteins, a suite of analytics probed docking solutions from the compound library, generating a ranked list of 100 compounds for each site. Based on factors inclusive of availability and low potential for toxicity, compounds were acquired and used in in-parallel selection and de-selection functional assays. Cancer cell growth inhibition was a positive selection criterion, with additional ranking based on greater potency and broader efficacy. Bone marrow toxicity is limiting for most drugs, and inhibition of bone marrow stem cell growth was as a deselection criterion. Multiple compounds clustered around 2 sites on 2 different proteins. One compound, Dxr2-017, exhibited very high relative efficacy against colon and breast cancer cells. Evaluation of Dxr2-017 in the NCI-60 panel corroborated these findings, and showed even higher efficacy against melanoma. We demonstrated that Dxr2-017 inhibited melanoma growth with low nanomolar efficacy, while concentrations of over 2300-fold higher had only limited inhibitory effects on bone marrow stem cells. Conclusions: This study provides proof-of-principle that protein structure constitutes high value primary information that can support identification of novel therapeutics. This approach is widely applicable and expandable. Citation Format: Fangfang Qiao, T. Andrew Binkowski, Wayne Anderson, Weining Chen, Gray Schiltz, Karl Scheidt, Amarnath Natarajan, Raymond Bergan. Protein structure inspired drug discovery [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 4493.

Suppression of B-Cell Activation by Human Cord Blood-Derived Stem Cells (CB-SCs) through the Galectin-9-Dependent Mechanism.

We developed the Stem Cell Educator therapy among multiple clinical trials based on the immune modulations of multipotent cord blood-derived stem cells (CB-SCs) on different compartments of immune cells, such as T cells and monocytes/macrophages, in type 1 diabetes and other autoimmune diseases. However, the effects of CB-SCs on the B cells remained unclear. To better understand the molecular mechanisms underlying the immune education of CB-SCs, we explored the modulations of CB-SCs on human B cells. CB-SCs were isolated from human cord blood units and confirmed by flow cytometry with different markers for their purity. B cells were purified by using anti-CD19 immunomagnetic beads from human peripheral blood mononuclear cells (PBMCs). Next, the activated B cells were treated in the presence or absence of coculture with CB-SCs for 7 days before undergoing flow cytometry analysis of phenotypic changes with different markers. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized to evaluate the levels of galectin expressions on CB-SCs with or without treatment of activated B cells in order to find the key galectin that was contributing to the B-cell modulation. Flow cytometry demonstrated that the proliferation of activated B cells was markedly suppressed in the presence of CB-SCs, leading to the downregulation of immunoglobulin production from the activated B cells. Phenotypic analysis revealed that treatment with CB-SCs increased the percentage of IgD+CD27- naïve B cells, but decreased the percentage of IgD-CD27+ switched B cells. The transwell assay showed that the immune suppression of CB-SCs on B cells was dependent on the galectin-9 molecule, as confirmed by the blocking experiment with the anti-galectin-9 monoclonal antibody. Mechanistic studies demonstrated that both calcium levels of cytoplasm and mitochondria were downregulated after the treatment with CB-SCs, causing the decline in mitochondrial membrane potential in the activated B cells. Western blot exhibited that the levels of phosphorylated Akt and Erk1/2 signaling proteins in the activated B cells were also markedly reduced in the presence of CB-SCs. CB-SCs displayed multiple immune modulations on B cells through the galectin-9-mediated mechanism and calcium flux/Akt/Erk1/2 signaling pathways. The data advance our current understanding of the molecular mechanisms underlying the Stem Cell Educator therapy to treat autoimmune diseases in clinics.

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

A Dual Role of SOX11 Expression in the Formation of T-Cell Acute Lymphoblastic Leukemia in Mouse Models

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 10%-15% of pediatric and 25% of adult ALL cases. Intensified chemotherapy and bone marrow transplants have improved the survival of T-ALL patients. However, around 20% of patients still suffer from primary resistant or relapsed T-ALL. Therefore, improving our knowledge of T-ALL biology and developing precision oncology therapeutics is essential for further progress in T-ALL treatment. SRY-related HMG-box 11 (SOX11) is a transcription factor that regulates the development and survival of multiple embryonic tissues and is crucial for the nervous system. SOX11 is expressed in a multitude of tumor types, where it has been associated with either increased or decreased cell proliferation, metastatic potential, and with either good or bad prognosis, depending on cancer type. Despite being absent in the T-cell lineage, SOX11 is expressed in a subset of T-ALLs, where it plays an unknown role. Based on having a malignantly restricted expression, we hypothesize that SOX11 might be oncogenic in T-ALL. Aim: Identify the role of aberrant SOX11 expression in (pre)leukemic T-cells. Methods: Using a conditional Rosa26-SOX11 overexpression mouse model, we generated mice with T-cell specific (using Lck-cre) SOX11 expression (SOX11 Lck). To assess the self-renewal potential of SOX11 Lck thymocytes, we performed thymic transplantations on sublethally irradiated mice expressing a different allele of CD45. To study SOX11 in a leukemic context, we crossed the SOX11 Lck mice with two established T-ALL mouse models that are either driven by the overexpression of Lmo2 (SOX11 Lck-Lmo2 CD2) or by the loss of the tumor suppressor Pten (SOX11/Pten Lck). An aging cohort of these T-ALL models was followed, and tumor samples were collected. Thymi of 8-weeks old mice were used to study the impact of SOX11 in a pre-leukemic context, and T-cell tumors arising in these models were also used for RNA-sequencing. Using a retroviral strategy, we overexpressed SOX11 in human cord blood stem cells, which we differentiated towards the T-cell lineage ex vivo by co-culturing them on OP9 stromal cells expressing Delta-like Notch ligand 4. Furthermore, we transduced Jurkat cells with a SOX11 expression vector, or with an empty vector, and injected the cells into immunocompromised mice to assess the effect of SOX11 on tumor growth in vivo. Results: In SOX11 Lck mice, we found a significant increase in the immature double-negative 3 (DN3) thymic fraction, with a concomitant decrease in the amount of more mature double-positive CD4 +CD8 + (DP) thymocytes (Fig. A). A possible explanation for this phenotype would be the establishment of a stem-like self-renewal program similar to the one driven by Lmo2. However, by performing thymic transplantations into sublethally irradiated mice, we concluded that SOX11 does not confer self-renewal properties to thymocytes. Additionally, we aged a cohort of SOX11 Lck mice and did not observe formation of T-cell malignancies up to 500 days of age. Given that the expression of SOX11 does not suffice to produce T-cell tumors, we generated the SOX11 Lck-Lmo2 CD2 and SOX11/Pten Lck T-ALL mouse models. In SOX11 Lck-Lmo2 CD2 mice, we found a synergistic effect of the co-expression of both genes on the expansion of DN3 thymocytes (Fig. A), which led to accelerated leukemia formation (Fig. B). By sequencing the RNA of pre-leukemic and leukemic DN3 cells, we observed that SOX11 is mostly regulating cell adhesion, morphology, and cell-cell interactions to promote this oncogenic phenotype in Lmo2-driven T-ALL. On the other hand, SOX11/Pten Lck mice suffered from delayed leukemogenesis (Fig. B), and this tumor suppressor role might be associated with the decrease in DP cells observed in the pre-leukemic stage. Lastly, we set out to validate these findings in a human context. The ex vivo differentiation of human hematopoietic stem cells toward the T-cell lineage showed that SOX11 strongly accelerates early commitment to double-negative T-cell precursors. Also, we observed that Jurkat T-ALL cells engraft faster in immunocompromised mice when expressing SOX11. Conclusions: By generating multiple models of SOX11 expression in the T-cell lineage, we showed that SOX11 impacts T-cell differentiation, both in mice and humans, and that it can promote or hinder T-ALL formation depending on the oncogenic driver.

Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34+ and CD34+CD38-CD45RA-CD90+ Cells

Hypoxic conditions preserve the multipotency and self-renewing capacity of murine bone marrow and human cord blood stem cells. Blood samples stored in sealed blood gas tubes become hypoxic as leukocytes metabolize and consume oxygen. Taken together, these observations suggest that peripheral blood stem cell (PBSC) samples stored under airtight conditions become hypoxic, and that the stem cells contained may undergo qualitative or quantitative changes. This study aimed to determine the effect of storage for 8 hours in a sealed system on PBSC samples. Granulocyte colony-stimulating factor-mobilized PBSC samples were collected prospectively from 9 patients with myeloma or amyloidosis prior to apheresis, followed by measurement of CO2, O2, hydrogen ion (pH), lactate, and glucose concentrations in the blood and immunophenotyping of stem cell and multipotent progenitor cell populations before and after 8 hours of storage in sealed blood collection tubes. Blood concentrations of O2 and glucose and pH measurements were significantly decreased, whereas concentrations of CO2 and lactate were significantly increased after storage. Significantly higher concentrations of CD34+ cells (552 ± 84 cells/106total nucleated cells [TNCs] versus 985 ± 143 cells/106TNCs; P=.03), CD34+CD38- cells (98 ± 32 cells/106TNCs versus 158 ± 52 cells/106TNCs; P=.03), CD34+CD38+ cells (444 ± 92 cells/106TNCs versus 789 ± 153 cells/106TNCs; P=.03), and CD34+CD38-CD45RA-CD90+ cells (55 ± 17 cells/106TNCs versus 89 ± 25 cells/106TNCs; P=.02) were detected after 8 hours of storage. The changes in concentrations of CD34+CD38+ cells and CD34+ cells were inversely associated with the change in glucose concentration (P=.003 and P < .001, respectively) and positively associated with the change in lactate concentration (P=.01 and P <.001, respectively) after 8 hours of airtight storage. Storage of PBSC samples in a sealed, airtight environment is associated with microenvironmental changes consistent with hypoxia and increased concentrations of immunophenotypically defined stem cells. These results may have clinical implications with regard to the collection and processing of stem cell products and warrant confirmation with functional and mechanistic studies.

Outcome of human umbilical cord blood stem cell transplantation (CBT) for acute myeloid leukemia in patients achieving first complete remission after one versus two induction courses: a study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

We compared transplantation outcomes of adult patients with AML that underwent cord blood transplantation (CBT) in CR1 following 1 versus 2 induction courses. Study included 325 patients, 243 (75%) with 1 and 82 (25%) with 2 induction courses. Engraftment was lower for patients achieving CR1 after 1 vs. 2 induction courses: 91% vs. 99% (p = 0.02). Incidence of acute GVHD was similar, 38% and 36% (p = 0.81), as was 2-year chronic GVHD at 23.4% and 27.5%, respectively (p = 0.65). Two-year non-relapse mortality (NRM), relapse incidence (RI), leukemia-free survival (LFS), overall survival (OS) and GVHD-free, relapse-free survival (GRFS) were not statistically different between patients achieving CR1 with 1 vs. 2 induction courses with 23% vs. 24% (p = 0.87), 25% vs. 30% (p = 0.4), 52% vs. 46% (p = 0.3), 59% vs. 50% (p = 0.2), and 44% vs. 41% (p = 0.66), respectively. Results were confirmed by multivariable analysis, NRM (hazard ratio (HR) = 1.1; 95% CI, 0.6-1.8, p = 0.7), RI (HR = 1.4; 95% CI, 0.9-2.3, p = 0.1), LFS (HR = 1.3; 95% CI, 0.9-1.8, p = 0.2), OS (HR = 1.3; 95% CI, 0.9-1.9, p = 0.1), and GRFS (HR = 1.1; 95% CI, 0.8-1.5, p = 0.5). Overall, outcomes of AML patients undergoing CBT in CR1 achieved after 1 or 2 induction courses are similar.

Outcome of Human Umbilical Cord Blood Stem Cell Transplantation (CBT) for Acute Myeloid Leukemia in Patients Achieving First Complete Remission after One Versus Two Induction Courses: A Study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

Background: Achieving a first complete remission (CR1) is an important prognostic factor for transplantation outcome. However, there are no data in the setting of cord blood transplantation (CBT) indicating whether the number of induction courses (1 or 2) needed to achieve CR1, is of prognostic significance. As CBT is advantageous for acute myelogenous leukemia (AML) patients (pts) with positive pre transplant measurable residual disease (MRD) (Milano F, NEJM 2016), it is conceivable that in the CBT setting, no difference in transplantation outcome will be observed between pts achieving CR1 after 1 or 2 inductions.Methods: Using the European Society for Blood and Marrow Transplantation (EBMT)/Acute Leukemia Working Party (ALWP) registry, we compared transplantation outcomes of adult pts aged ≥18 years with AML that underwent CBT in 2005-2020 in CR1, achieved following 1 versus (vs) 2 induction courses. Multivariate analysis (MVA) adjusting for differences between the induction groups was performed using a Cox's proportional-hazards regression model for main outcomes.Results: Three hundred and twenty-five pts were included comprising 243 (75%) with 1 and 82 (25%) with 2 induction chemotherapy courses. Median (range) follow-up was 65.4 (57.4-73.5) and 51.0 (34.8-61.5) months, respectively (p=0.6). Median age was 49.4 (19.0-70.9) and 52.1 (19.2-71.5) years (p=0.8), respectively. For patients with 1 and 2 induction courses, respectively, 49.4% and 57.3% were male, 225 (92.6%) and 78 (95.1%) pts had de novo AML, and 18 (7.4%) and 4 (4.9%) had secondary AML (p=0.6). Pts with 1 and 2 induction courses, respectively, were classified by cytogenetic risk as follows: intermediate, 62.0% and 79.2%, adverse, 33.2% and 19.5%, and favorable, 4.8% and 1.3% (p=0.02) (missing data~25%). The FLT3-ITD mutation was harbored by 33.7% and 32.3% of the pts (p=0.8), respectively (missing data ~6%). Conditioning was myeloablative (MAC) in 43.0% and 36.6% and reduced intensity (RIC) in 57.0% and 63.4%, respectively (p=0.31). Karnofsky performance score (KPS) was > 90 in 74.7% and 71% of the pts, respectively (p=0.5). The most frequent anti-graft-versus-host disease (GVHD) prophylaxis was cyclosporin A (CSA) and mycophenolate mofetil (MMF) in 75.6% and 82.5 %, or CSA with or without steroids in 16.1 % and 11.2%, respectively. Anti-thymocyte globulin (ATG) was administered to 32.9% and 25.6% of the CBT recipients, respectively (p=0.2). Engraftment rates were lower for pts achieving CR1 after 1 vs 2 induction courses (91.3% and 98.8% p=0.02) with corresponding day 60 absolute neutrophil count (ANC) > 0.5 x 10 9/L in 89.6% vs 96.3% of the pts (p=0.03).Day 180 incidence of acute GVHD grades II-IV was similar in both induction groups, 38.3% and 37.2% (p=0.8), as was 2-year total chronic GVHD, 23.4% and 27.5 %, respectively (p=0.6). In univariate analysis, the 2–year non-relapse mortality (NRM), relapse incidence (RI), leukemia-free survival (LFS), overall survival (OS) and GVHD-free, relapse-free survival (GRFS) were similar between patients achieving CR1 with 1 vs 2 induction courses with 22.6% vs 23.6% (p=0.87) 25.1% vs 30.4% (p=0.4), 52.3% vs 46.0% (p=0.3), 58.6% vs 50.0% (p=0.2), and 44% vs 44.1% (p=0.66), respectively. Similarly in MVA, there was no significant association between 2 courses of induction and NRM (hazard ratio (HR) = 1.1; 95% CI, 0.6-1.8, p=0.7), RI (HR = 1.4; 95% CI, 0.9-2.3, p=0.1), LFS (HR = 1.3; 95% CI, 0.9-1.8, p=0.2), OS (HR = 1.3; 95% CI, 0.9-1.9, p=0.1), and GRFS (HR = 1.1; 95% CI, 0.8-1.5, p=0.5).Conclusions: In CBT recipients, we did not find any significant differences in outcomes in patients achieving CR1 after one or two induction courses. Notably, engraftment was better in patients receiving two courses of induction chemotherapy. [Display omitted] DisclosuresLabopin: Jazz Pharmaceuticals: Honoraria. Forcade: Novartis: Other: travel grant. Sierra: Amgen: Other: Educational grant; Roche: Other: Educational grant; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion: Other: Educational grant; Jazz Pharmaceuticals: Research Funding; Janssen: Other: Educational grant; Novartis: Honoraria, Research Funding, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria; BMS Celgene: Honoraria, Research Funding. Byrne: Incyte: Honoraria. Blaise: Jazz Pharmaceuticals: Honoraria. Mohty: Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Adaptive Biotechnologies: Honoraria.

Therapeutic use of red blood cells and platelets derived from human cord blood stem cells.

Red blood cells (RBCs) and platelets derived from stem cells are possible solutions to the increasing demand for blood transfusion. Based on the availability of stem cells, their relatively defined differentiation mechanisms, and the massive exploration of induction systems, the generation of RBCs or platelets in vitro from cord blood hematopoietic stem/progenitor cells (CB‐HSPCs) has potential for clinical applications. However, information on the clinical translation of stem cell‐derived RBCs and platelets in the literature and at the ClinicalTrials.gov website is very limited. The only clinical trial on cultured RBCs, which aimed to assess the lifespan of RBCs cultured in vivo, was reported by Luc Douay and colleagues. Of note, the cultured RBCs they used were derived from autologous peripheral blood HSPCs, and no cultured platelets have been applied clinically to date. However, CB‐HSPC‐derived megakaryocytes, platelet precursors, have been used in the treatment of thrombocytopenia. A successful phase I trial was reported, followed by phase II and III clinical trials conducted in China. In this review, the gap between the many basic studies and limited clinical trials on stem cell‐derived RBCs and platelets is summarized. The possible reasons and solutions for this gap are discussed. Further technological improvements for blood cell expansion and maturation ex vivo and the establishment of biological standards for stem cell derivatives might help to facilitate the therapeutic applications of cultured RBCs and platelets derived from CB‐HSPCs in the near future.

Effect of umbilical cord blood stem cell transplantation on restenosis after endovascular interventional therapy for diabetic hindlimb vascular disease.

This study aimed to investigate the mechanism of human umbilical cord blood stem cell (HUCBSC) transplantation on restenosis after percutaneous transluminal angioplasty (PTA) for diabetic hindlimb vascular disease in rabbits. After successfully preparing a rabbit model of diabetic hindlimb vascular disease, 16 rabbits were randomly assigned to two groups. Of these, 8 rabbits received PTA surgery alone (PTA group), and the other 8 rabbits received PTA and HUCBSC (PTA+HUCBSC group) treatments. Five more healthy rabbits were set as healthy control (HC group). Samples were collected after 4 weeks of treatment. The expressions of regulator of calcineurin 1 (RCAN1) and calcineurin A (CnA) in the diseased artery were detected by immunofluorescence staining. The distribution of HUCBSCs was observed by pathological examination in transplanted artery, distal artery, and liver. Cytology experiments were applied to assess the levels of JAK and STAT3, and the migration and proliferation of human aortic vascular smooth muscle cells (HA-VSMC). In the rabbit model of diabetic vascular lesions in the hindlimbs, we found the stenosis of the femoral artery became more and more serious with time, and the expression level of PCNA positive cells was also gradually increased. The expression levels of RCAN1 and CnA in the PTA+HUCBSC group were significantly lower than those in PTA group. HUCBSC inhibited the migration and proliferation of HA-VSMC via JAK/STAT3 pathway. After HUCBSC local transplantation, HUCBSC had no distal tissue distribution. HUCBSC transplantation may prevent restenosis after PTA of diabetic hindlimb vascular disease through JAK/STAT3 pathway.

Transport study of interleukin-1 inhibitors using a human in vitro model of the blood-brain barrier

The proinflammatory cytokine Interleukin-1 (IL-1), with its two isoforms α and β, has important roles in multiple pathogenic processes in the central nervous system. The present study aimed to evaluate and compare the blood-to-brain distribution of anakinra (IL-1 receptor antagonist), bermekimab (IL-1α antagonist) and canakinumab (IL-1β antagonist).A human in vitro model of the blood-brain barrier derived from human umbilical cord blood stem cells was used, where isolated CD34+ cells co-cultured with bovine pericytes were matured into polarized brain-like endothelial cells. Transport rates of the three test items were evaluated after 180 ​min incubation at concentrations 50, 250 and 1250 ​nM in a transwell system. We report herein that anakinra passes the human brain-like endothelial monolayer at a 4-7-fold higher rate than the monoclonal antibodies tested. Both antibodies had similar transport rates at all concentrations. No dose-dependent effects in transport rates were observed, nor any saturation effects at supraphysiological concentrations. The larger propensity of anakinra to pass this model of the human blood-brain barrier supports existing data and confirms that anakinra can reach the brain compartment at clinically relevant concentrations. As anakinra inhibits the actions of both IL-1α and IL-1β, it blocks all effects of IL-1 downstream signaling. The results herein further add to the growing body of evidence of the potential utility of anakinra to treat neuroinflammatory disorders.

Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation.

Droplet vitrification has emerged as a promising ice‐free cryopreservation approach to provide a supply chain for off‐the‐shelf cell products in cell therapy and regenerative medicine applications. Translation of this approach requires the use of low concentration (i.e., low toxicity) permeable cryoprotectant agents (CPA) and high post cryopreservation viability (>90%), thereby demanding fast cooling and warming rates. Unfortunately, with traditional approaches using convective heat transfer, the droplet volumes that can be successfully vitrified and rewarmed are impractically small (i.e., 180 picoliter) for <2.5 m permeable CPA. Herein, a novel approach to achieve 90–95% viability in micro‐liter size droplets with 2 m permeable CPA, is presented. Droplets with plasmonic gold nanorods (GNRs) are printed onto a cryogenic copper substrate for improved cooling rates via conduction, while plasmonic laser heating yields >400‐fold improvement in warming rates over traditional convective approach. High viability cryopreservation is then demonstrated in a model cell line (human dermal fibroblasts) and an important regenerative medicine cell line (human umbilical cord blood stem cells). This approach opens a new paradigm for cryopreservation and rewarming of dramatically larger volume droplets at lower CPA concentration for cell therapy and other regenerative medicine applications.

Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one‑carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient's blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient's blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these "educated" immune cells to the patient's blood. Exosomes are nano-sized extracellular vesicles between 30‒150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Human immune cells infiltrate the spinal cord and impair recovery after spinal cord injury in humanized mice

Humanized mice can be used to better understand how the human immune system responds to central nervous system (CNS) injury and inflammation. The optimal parameters for using humanized mice in preclinical CNS injury models need to be established for appropriate use and interpretation. Here, we show that the developmental age of the human immune system significantly affects anatomical and functional outcome measures in a preclinical model of traumatic spinal cord injury (SCI). Specifically, it takes approximately 3–4 months for a stable and functionally competent human immune system to develop in neonatal immune compromised mice after they are engrafted with human umbilical cord blood stem cells. Humanized mice receiving a SCI before or after stable engraftment exhibit significantly different neuroinflammatory profiles. Importantly, the development of a mature human immune system was associated with worse lesion pathology and neurological recovery after SCI. In these mice, human T cells infiltrate the spinal cord lesion and directly contact human macrophages. Together, data in this report establish an optimal experimental framework for using humanized mice to help translate promising preclinical therapies for CNS injury.

Distinct Mechanisms Underlying the Ex Vivo Expansion of Human Cord Blood Stem Cells with Different Strategies Currently Used for Allogeneic Transplantation

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSCs) for patients that require allogeneic stem cell transplantation. The use of UCB in adults is restricted due to the limited number of HSCs within a single unit. This limitation can be overcome by ex-vivo expansion. Several strategies have successfully expanded the numbers of clinically functional HSCs. Such strategies, including the aryl hydrocarbon receptor antagonist, SR1, pyrimidoindole derivative, UM171, nicotinamide, NAM and more recently, valproic acid (VPA), have entered clinical development. HSC grafts expanded by SR1 and UM171 have been reported to establish rapid and sustained hematological reconstitution in allogeneic transplant recipients [1-3]. Our group has started a clinical trial using VPA-expanded allogeneic UCB grafts (NCT03885947). Initial data have shown multi-lineage donor cell reconstitution with especially rapid T cell and platelet engraftment in patients with treatment refractory acute leukemias. To gain insight into the mechanisms underlying these clinical HSC expansion procedures, we recreated the different approaches to compare the various expanded grafts. Although there is successful clinical data with the NAM approach, we were unable to recreate these conditions in our laboratory and therefore, we could not compare these grafts to those expanded with SR1, UM171 and VPA [4-7]. These comparative studies indicated that each strategy increased to varying degrees both the percentage and absolute numbers of CD34+CD133+CD90+EPCR+ as opposed to cultures treated with their respective cytokine cocktail alone. Although SR1 treatment resulted in the generation of greater numbers of total nucleated cells from purified 1x106 UCB-CD34+ cells (16x107 in SR1 vs 8x107 in UM171 and 7x107 in VPA), the numbers of CD34+CD133+CD90+EPCR+ cells (4x105 in SR1 vs, 7x105 in UM171 and 8.7x105 in VPA) as well as of other CD34+ subpopulations with different degrees of stem cell activity were lower than those observed in VPA and UM171-expanded cultures. In order to understand the underlying mechanism by which these agents act, we isolated CD34+CD90-EPCR- cells and treated with VPA, SR1 or UM171. VPA treatment for 24 hrs resulted in acquisition of CD90 and EPCR expression (80% and 25% of cells acquired CD90 and EPCR, respectively). UM171 treatment led to a modest increase in cells expressing EPCR and CD90. By contrast, SR1 treatment did not induce a phenotypic switch. Comparison of RNA-seq data indicated a strong induction of CD90, EPCR and CD133 in the CD34+ grafts expanded with VPA and UM171, but not in grafts generated with SR1. Twenty five out of 53 and 5 out of 9 genes upregulated more than 2 fold by UM171 and SR1, respectively, were also upregulated in VPA-expanded grafts. Eight out of 39 and 6 out of 77 genes downregulated more than 1.5 fold by UM171 and SR1, respectively, were also downregulated in VPA-expanded grafts. However, grafts expanded with each of these agents shared only three upregulated and three downregulated genes suggesting that different mechanisms underlie the actions of each of these agents. In fact, enhanced histone acetylation induced by VPA was not observed in SR1 and UM171 expanded cells. Both UM171 and VPA did not signal through the AHR pathway, which is required for SR1-mediated expansion. These data suggest that these agents target distinct pathways leading to ex vivo HSC expansion. Since mitochondrial activity is an additional critical regulator of the HSC fate, we examined the mitochondrial profile. VPA-expanded HSCs exhibited a remodeled mitochondrial network associated with low levels of mitochondrial ROS and mass. HSCs expanded with UM171 unlike SR1 displayed low ROS levels. Neither UM171 nor SR1 impacted mitochondrial mass. In vivo transplantation of UM171 and VPA expanded grafts in NSG mice showed similar degrees of human cell chimerism (~25%). Whereas both expanded grafts were capable of establishing multilineage hematopoiesis, we found a greater degree of CD34+ and T cell chimerism in NSG mice engrafted with VPA-expanded grafts. Studies of the engraftment potential of SR1 expanded grafts are ongoing. In conclusion, our findings suggest that although VPA, UM171 and SR1 expand greater numbers of human HSCs that are useful for allogeneic stem cell transplantation, their underlying mechanisms of action and pathways involved are different. Disclosures Schaniel: Summer Road: Research Funding. Hoffman:Merus: Research Funding.

Donor-to-Donor Heterogeneity in the Clonal Dynamics of Transplanted Human Cord Blood Stem Cells in Murine Xenografts.

Umbilical cord blood (UCB) provides an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Administration of sufficient donor HSCs is critical to restore recipient hematopoiesis and to maintain long-term polyclonal blood formation. However, due to lack of unique markers, the frequency of HSCs among UCB CD34+ cells is the subject of ongoing debate, urging for reproducible strategies for their counting. Here, we used cellular barcoding to determine the frequency and clonal dynamics of human UCB HSCs and to determine how data analysis methods affect these parameters. We transplanted lentivirally barcoded CD34+ cells from 20 UCB donors into Nod/Scid/IL2Ry-/- (NSG) mice (n = 30). Twelve recipients (of 8 UCB donors) engrafted with >1% GFP+ cells, allowing for clonal analysis by multiplexed barcode deep sequencing. Using multiple definitions of clonal diversity and strategies for data filtering, we demonstrate that differences in data analysis can change clonal counts by several orders of magnitude and propose methods to improve their consistency. Using these methods, we show that the frequency of NSG-repopulating cells was low (median ∼1 HSC/104 CD34+ UCB cells) and could vary up to 10-fold between donors. Clonal patterns in blood became increasingly consistent over time, likely reflecting initial output of transient progenitors, followed by long-term HSCs with stable hierarchies. The majority of long-term clones displayed multilineage output, yet clones with lymphoid- or myeloid-biased output were also observed. Altogether, this study uncovers substantial interdonor and analysis-induced variability in the frequency of UCB CD34+ clones that contribute to post-transplant hematopoiesis. As clone tracing is increasingly relevant, we urge for universal and transparent methods to count HSC clones during normal aging and upon transplantation.

A Gutsy Move for Cell-Based Regenerative Medicine in Parkinson\u2019s Disease: Targeting the Gut Microbiome to Sequester Inflammation and Neurotoxicity

Pharmaceuticals and cell-based regenerative medicine for Parkinson’s disease (PD) offer palliative relief but do not arrest the disease progression. Cell therapy has emerged as an experimental treatment, but current cell sources such as human umbilical cord blood (hUCB) stem cells display only partial recapitulation of mature dopaminergic neuron phenotype and function. Nonetheless, stem cell grafts ameliorate PD-associated histological and behavioral deficits likely through stem cell graft-secreted therapeutic substances. We recently demonstrated the potential of hUCB-derived plasma in enhancing motor capabilities and gastrointestinal function, as well as preventing dopaminergic neuronal cell loss, in an 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) rodent model of PD. Recognizing the translational need to test in another PD model, we now examined here the effects of an intravenously transplanted combination of hUCB and plasma into the 6-hydroxydopamine (6-OHDA) lesioned adult rats. Animals received three separate doses of 4 × 106 hUCB cells with plasma beginning at 7 days after stereotaxic 6-OHDA lesion, then behaviorally and immunohistochemically evaluated over 56 days post-lesion. Whereas vehicle-treated lesioned animals exhibited the typical 6-OHDA neurobehavioral symptoms, hUCB and plasma-treated lesioned animals showed significant attenuation of motor function, gut motility, and nigral dopaminergic neuronal survival, combined with diminished pro-inflammatory microbiomes not only in the nigra, but also in the gut. Altogether these data support a regenerative medicine approach for PD by sequestering inflammation and neurotoxicity through correction of gut dysbiosis.

Effect of intracameral human cord blood-derived stem cells on lasered rabbit trabecular meshwork.

This study aimed to investigate the effect of intracameral human cord blood stem cells on lasered rabbit trabecular meshwork. Immediately following diode laser application to the trabecular meshwork, human cord blood stem cells were injected intracamerally, in one eye of 12 albino rabbits. The other eye of ten rabbits was lasered controls and two eyes were normal controls. Rabbits were killed after 4, 8 and 12weeks. Lasered control rabbit eyes showed significant disruption of trabecular architecture, loss and pleomorphism of trabecular endothelial cells and progressive narrowing of trabecular spaces till 12weeks. In contrast, lasered eyes, concurrently injected with human cord blood stem cells, showed relatively preserved endothelial cellularity and structure of the trabecular meshwork, at all time points. Human CD34- and CD44-positive cells were identified in 7/8 eyes treated with stem cells, at 4 and 8weeks, and 2 of 3 at 12weeks. Many PKH26-labeled human cord blood cells were visible throughout the trabecular area at 4weeks. They gradually decreased in number by 8weeks, and at 12weeks, they appeared to be oriented along trabecular beams. There was a relative preservation of cellularity and architecture of the trabecular meshwork in eyes injected with human cord blood stem cells, as compared to lasered control eyes up to 12weeks, without significant inflammation. This suggests a probable role for such stem cells in eyes with glaucoma, having trabecular dysfunction.

Cyclosporine May Inhibit the Effect of Extra-Physiologic Oxygen Shock/Stress on Peripheral Blood Stem Cells

Introduction: Adequate numbers of stem cells with preserved multi-potency and self-renewing capabilities are necessary for successful hematopoietic reconstitution after bone marrow transplantation. Although hematopoietic stem cells (HSC) reside in the bone marrow under a hypoxic microenvironment (1-4% O2), human HSC are collected and processed in ambient air (21% O2) (Spencer et al., Nature 508:269-73). Exposure of murine bone marrow and human cord blood to ambient air for as little as 30 minutes triggered stem cell differentiation from quiescent pluripotent long term stem cells into activated multipotent progenitors (MPPs), a phenomenon called extra-physiologic oxygen shock / stress (EPHOSS)(Mantel et al., Cell 161:1553-65). The effect of EPHOSS on HSCs is mediated through reactive oxygen species (ROS) which open the mitochondrial permeability transition pore (MPTP) and trigger stem cell differentiation. Cyclosporine (CSA) inhibits the MPTP regulator cyclophilin D and prevents MPTP opening (Kroemer et al., Physiol Rev 87:99-163). Currently, mobilized peripheral blood stem cells (PBSCs) are the major source of grafts for hematopoietic cell transplantation. We hypothesized that EPHOSS is detrimental to human PBSCs similar to murine bone marrow and human cord blood stem cells and CSA will protect human PBSCs from the effects of EPHOSS and inhibit their differentiation from pluripotent long term HSC into short term MPPs.Study design and methods. This is a proof-of-concept, prospective, non-interventional study. We obtained G-CSF mobilized PBSCs from healthy related donors under an IRB approved protocol. All donors provided written informed consent. Blood containing HSCs was collected from each donor with minimal exposure to ambient air (<60 seconds). The sample was immediately split and incubated for 60 minutes with and without CSA (50 µg/mL). CSA treated and untreated PBSCs were immunophenotyped by multi-parameter flow cytometry for HSCs (CD34+CD38-CD90+CD45RA-), MPP (CD34+CD38-CD90-CD45RA-) and common lymphoid progenitors (CD34+CD38+CD127+). We used a CD34 ISHAGE-based gating strategy to accurately enumerate HSC and MPP.Results. In four separate experiments, CSA treated PBSCs had a higher median (range) number of HSCs/106 total nucleated cells (TNC) compared to untreated PBSCs ((297 (51 - 512) versus 185 (47 -392), respectively). CSA treated PBSCs also had a higher median (range) HSC:MPP ratio compared to untreated PBSCs ((0.56 (0.40 - 0.70) versus 0.43 (0.30 - 0.50), respectively) suggesting that the differentiation of HSC to MPP upon exposure to air was decreased in the CSA treated PBSCs.Conclusions / future directions. This is the first report describing the effect of EPHOSS in human PBSCs. Our preliminary data suggest that EPHOSS promotes the differentiation of human PBSCs from HSC to MPP and that CSA may inhibit this process. Further confirmatory and mechanistic studies exploring the contribution of MPTP and ROS to EPHOSS are ongoing. The results from this research may potentially change the current practice of collecting PBSCs in ambient air. DisclosuresMcCarthy:Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Chen:Bellicum Pharmaceuticals: Research Funding.