Alternative Stem Cell Source Research Articles

A small-molecule combination promotes ex vivo expansion of cord blood-derived hematopoietic stem cells

Background & Aim Background Cord blood (CB) provides an excellent alternative source of hematopoietic stem cells (HSCs) for patients lacking human leukocyte antigen-matched peripheral blood or bone marrow graft for transplantation. However, the limited number of HSCs in CB graft restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for HSCs expansion and self-renewal. And the effect of small molecules on the proliferation and differentiation of HSCs has not been reported at the single cell level. Here, we report a combination including the small molecules UM171, SR1, JNK-IN-8 (USK) and the cytokines TPO, IL-6, SCF, FLT-3L, which can efficiently promote ex vivo expansion of HSCs. Methods, Results & Conclusion Methods CB-derived CD34+ cells were cultured for 10 days with cytokines plus DMSO (Vehicle group) or with cytokines plus three small molecules (USK group). The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. Results USK treatment increased the proportion of CD34+ (92.29 ±2.54%), CD34+ CD38− cells (78.06 ± 5.52%) and CD34+CD38−CD45RA−CD90+ LT-HSCs (8.96 ± 2.83%) compared to vehicle treatment (52.57 ± 5.98%, 39.31 ± 4.62% and 0.80 ± 0.54%, respectively, n=5). USK treatment led to a 106.52-, 923.8-, and 603.04-fold increase in CD34+, CD34+ CD38− and LT-HSCs numbers relative to initial cells, respectively (n=5). Vehicle treatment led to a 72.82-, 508.81-, and 54.64-fold increase in CD34+, CD34+ CD38− and LT-HSCs numbers relative to initial cells, respectively (n=5). USK treatment led to an 11.04-fold increase in LT-HSC numbers as compared to vehicle cultures. Conclusions Our study shows that a small-molecule combination (USK) can efficiently promote ex vivo expansion of HSCs. Using this combination model combined with transcriptome analysis at the single cell level, the key pathways and molecules of hematopoietic stem cell self-renewal in vitro can be further studied, which is expected to provide new targets for in vitro expansion of hematopoietic stem cells.

Human Wharton's Jelly-Cellular Specificity, Stemness Potency, Animal Models, and Current Application in Human Clinical Trials.

Stem cell therapies offer a great promise for regenerative and reconstructive medicine, due to their self-renewal and differentiation capacity. Although embryonic stem cells are pluripotent, their utilization involves embryo destruction and is ethically controversial. Therefore, adult tissues that have emerged as an alternative source of stem cells and perinatal tissues, such as the umbilical cord, appear to be particularly attractive. Wharton’s jelly, a gelatinous connective tissue contained in the umbilical cord, is abundant in mesenchymal stem cells (MSCs) that express CD105, CD73, CD90, Oct-4, Sox-2, and Nanog among others, and have the ability to differentiate into osteogenic, adipogenic, chondrogenic, and other lineages. Moreover, Wharton’s jelly-derived MSCs (WJ-MSCs) do not express MHC-II and exhibit immunomodulatory properties, which makes them a good alternative for allogeneic and xenogeneic transplantations in cellular therapies. Therefore, umbilical cord, especially Wharton’s jelly, is a promising source of mesenchymal stem cells.

A comparison of the use of adipose-derived and bone marrow-derived stem cells for peripheral nerve regeneration in vitro and in vivo

BackgroundTo date, it has repeatedly been demonstrated that infusing bone marrow-derived stem cells (BMSCs) into acellular nerve scaffolds can promote and support axon regeneration through a peripheral nerve defect. However, harvesting BMSCs is an invasive and painful process fraught with a low cellular yield.MethodsIn pursuit of alternative stem cell sources, we isolated stem cells from the inguinal subcutaneous adipose tissue of adult Sprague–Dawley rats (adipose-derived stem cells, ADSCs). We used a co-culture system that allows isolated adult mesenchymal stem cells (MSCs) and Schwann cells (SCs) to grow in the same culture medium but without direct cellular contact. We verified SC phenotype in vitro by cell marker analysis and used red fluorescent protein-tagged ADSCs to detect their fate after being injected into a chemically extracted acellular nerve allograft (CEANA). To compare the regenerative effects of CEANA containing either BMSCs or ADSCs with an autograft and CEANA only on the sciatic nerve defect in vivo, we performed histological and functional assessments up to 16 weeks after grafting.ResultsIn vitro, we observed reciprocal beneficial effects of ADSCs and SCs in the ADSC–SC co-culture system. Moreover, ADSCs were able to survive in CEANA for 5 days after in vitro implantation. Sixteen weeks after grafting, all results consistently showed that CEANA infused with BMSCs or ADSCs enhanced injured sciatic nerve repair compared to the acellular CEANA-only treatment. Furthermore, their beneficial effects on sciatic injury regeneration were comparable as histological and functional parameters evaluated showed no statistically significant differences. However, the autograft group was roundly superior to both the BMSC- or ADSC-loaded CEANA groups.ConclusionThe results of the present study show that ADSCs are a viable alternative stem cell source for treating sciatic nerve injury in lieu of BMSCs.

Gene expression profiling of progenitor cells isolated from rat rotator cuff musculotendinous junction

BackgroundRotator cuff tendon tears are typically degenerative and usually affect the region of tendon insertion on bone. The remnant torn tendon is degenerative and may not be an ideal source for progenitor cells for cell-based therapies. Therefore, the aim of this study was to determine if musculotendinous junction (MTJ), which is adjacent to tendon would be a viable alternate source of progenitor stem cells. We also sought to study the gene expression profile MTJ progenitors and compare it with progenitors isolated from RC tendon, RC muscle and other existing tissue sources (bone marrow, adipose tissue, and Achilles tendon).MethodsRotator cuff tendon (RCT), muscle (RCM), and RCMTJ as well as Achilles tendon (AT) tissues were harvested from healthy male Lewis rats and progenitor cultures were established from these tissues and also from bone marrow and adipose tissue. Quantitative RT-PCR was performed on RNA extracts from intact tissues and progenitor cells using a custom array for the mesenchymal stem cell (MSC) differentiation marker genes. The gene expression profile of MSC differentiation markers within four tissues types, six progenitor cells, and between tissue and their corresponding progenitors were compared.ResultsProgenitors cells can be isolated from rat rotator cuff musculotendinous tissue and their pattern of MSC gene expression was similar to the rotator cuff tendon progenitors for majority of the genes tested. However, there were significant differences between the MSC gene expression patterns of RCMTJ and RCM progenitors. Furthermore, there were differences in gene expression between the RCMTJ tissue and its progenitor cells with respect to MSC differentiation markers. The gene expression pattern of RCMTJ tissue was similar to RCM tissue with respect to markers of chondrogenesis, myogenesis, tenogenesis, and MSC specific markers.ConclusionWe demonstrate that the musculotendinous junction contains distinct set of progenitor cells and their MSC gene expression pattern is similar to rotator cuff tendon progenitors. RCMTJ progenitors will be an attractive option for cell-based regenerative treatment of chronic rotator cuff tears.

Alternative donors provide comparable results to matched unrelated donors in patients with acute lymphoblastic leukemia undergoing allogeneic stem cell transplantation in second complete remission: a report from the EBMT Acute Leukemia Working Party.

Relapse of acute lymphoblastic leukemia (ALL) remains a major therapeutic challenge. Despite the consensus for proceeding to allogeneic stem cell transplantation (HSCT) in relapsing patients with ALL who achieve second complete remission (CR2) with salvage therapy, most patients lack a suitable matched-related histocompatible donor. The present multicenter retrospective study compared, for ALL patients in CR2, the HSCT outcome from all four possible alternative hematopoietic stem cell sources, namely matched unrelated 10/10 (n = 281), mismatched unrelated 9/10 (n = 125), haploidentical (n = 105), and cord blood (n = 104) donors. The 2-year outcomes were not statistically different between the four donor sources with respect to overall survival (38.3-47.2%), leukemia-free survival (30.5-39.6%), relapse incidence (32.6-37.6%), nonrelapse mortality (27.5-34.6%), and graft-versus-host disease-free relapse survival (21.4-33.1%). Donor choices for ALL patients achieving CR2 post first relapse are broad, ensuring that most patient in need secures a graft. Therefore, in practice, the donor choice should depend on timely availability and policy center.

Prospective evaluation of alternative donor from unrelated donor and cord blood in adult acute leukemia and myelodysplastic syndrome.

A prospectively registered observational study was conducted to assess the significance of allogeneic hematopoietic stem cell transplantation from highly HLA-matched unrelated donors (UD) and cord blood (CB) on outcomes in adult acute leukemia (AL) and myelodysplastic syndrome (MDS). Between 2007 and 2015, 231 transplant-eligible patients were registered for a phase 2 study of alternative donor transplantation. After registration, a sufficient time period was given to find appropriate UD. Patients received CB transplantation (CBT) if an appropriate UD was unavailable. In total, 119 patients received CBT (106 AL and 13 MDS) and 91 patients received UD transplantation (UDT) (86 AL and 5 MDS). The median age was 39 years in both groups. The primary objective was overall survival (OS); secondary objectives included cumulative incidences of non-relapse mortality (NRM) and relapse, and disease-free survival. Diagnosis, disease status at transplantation, refined disease risk index, and hematopoietic cell transplant-specific comorbidity index did not differ between UDT and CBT. In multivariate analyses, graft source was not a significant risk factor for all objectives. In adjusted analyses, UDT and CBT showed similar OS, NRM, and relapse in this prospective study. CB can be a comparable alternative stem cell source to UD by achieving a timely transplant.

Research progresses of the way to improve umbilical cord blood stem cells homing and engraftment

Umbilical cord blood is an alternative hematopoietic stem cell source has been widely recognized. Initially, umbilical cord blood transplantation was limited, given the low engraftment. So the method of improving cord blood homing and engraftment has been widely studied in various fields. This paper briefly reviews the progress of main researches in recent years. Key words: Cord blood stem cell transplantation; Homing; Engraftment; Review

Generation of human androgenetic induced pluripotent stem cells

In humans, parthenogenesis and androgenesis occur naturally in mature cystic ovarian teratomas and androgenetic complete hydatidiform moles (CHM), respectively. Our previous study has reported human parthenogenetic induced pluripotent stem cells from ovarian teratoma–derived fibroblasts and screening of imprinted genes using genome-wide DNA methylation analysis. However, due to the lack of the counterparts of uniparental cells, identification of new imprinted differentially methylated regions has been limited. CHM are inherited from only the paternal genome. In this study, we generated human androgenetic induced pluripotent stem cells (AgHiPSCs) from primary androgenetic fibroblasts derived from CHM. To investigate the pluripotency state of AgHiPSCs, we analyzed their cellular and molecular characteristics. We tested the DNA methylation status of imprinted genes using bisulfite sequencing and demonstrated the androgenetic identity of AgHiPSCs. AgHiPSCs might be an attractive alternative source of human androgenetic embryonic stem cells. Furthermore, AgHiPSCs can be used in regenerative medicine, for analysis of genomic imprinting, to study imprinting-related development, and for disease modeling in humans.

Mesenchymal stem cells derived from iPSCs expressing interleukin-24 inhibit the growth of melanoma in the tumor-bearing mouse model

BackgroundInterleukin-24 (IL-24) is a therapeutic gene for melanoma, which can induce melanoma cell apoptosis. Mesenchymal stem cells (MSCs) show promise as a carrier to delivery anti-cancer factors to tumor tissues. Induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells (MSCs). We previously developed a novel non-viral gene targeting vector to target IL-24 to human iPSCs. This study aims to investigate whether MSCs derived from the iPSCs with the site-specific integration of IL-24 can inhibit the growth of melanoma in a tumor-bearing mouse model via retro-orbital injection.MethodsIL-24-iPSCs were differentiated into IL-24-iMSCs in vitro, of which cellular properties and potential of differentiation were characterized. The expression of IL-24 in the IL-24-iMSCs was measured by qRT-PCR, Western Blotting, and ELISA analysis. IL-24-iMSCs were transplanted into the melanoma-bearing mice by retro-orbital intravenous injection. The inhibitory effect of IL-24-iMSCs on the melanoma cells was investigated in a co-culture system and tumor-bearing mice. The molecular mechanisms underlying IL-24-iMSCs in exerting anti-tumor effect were also explored.ResultsiPSCs-derived iMSCs have the typical profile of cell surface markers of MSCs and have the ability to differentiate into osteoblasts, adipocytes, and chondroblasts. The expression level of IL-24 in IL-24-iMSCs reached 95.39 ng/106 cells/24 h, which is significantly higher than that in iMSCs, inducing melanoma cells apoptosis more effectively in vitro compared with iMSCs. IL-24-iMSCs exerted a significant inhibitory effect on the growth of melanoma in subcutaneous mouse models, in which the migration of IL-24-iMSCs to tumor tissue was confirmed. Additionally, increased expression of Bax and Cleaved caspase-3 and down-regulation of Bcl-2 were observed in the mice treated with IL-24-iMSCs.ConclusionMSCs derived from iPSCs with the integration of IL-24 at rDNA locus can inhibit the growth of melanoma in tumor-bearing mouse models when administrated via retro-orbital injection.

Seven Years of Haplo-Cord Transplantation: Immune Reconstitution and Outcomes Using Anti-Thymocyte Globulin

Introduction Haplo-cord transplantation – the co-infusion of a single cord unit with CD34-selected cells from a haplo-identical donor, provides an alternate stem cell source for patients lacking HLA-matched donors. Anti-thymocyte globulin (ATG) is routinely used in haplo-cord preparative regimens to reduce graft failure, graft vs. host disease (GVHD) and graft vs. graft reactions. However, excessive depletion of cord lymphocytes may compromise immune reconstitution and graft vs. tumor (GVT) effects. Methods We reviewed lymphocyte recovery and outcome data for consecutive patients with hematologic malignancy who underwent haplo-cord transplantation at Weill Cornell Medicine between June 2012 and June 2019. All were conditioned using fludarabine and melphalan with or without total body irradiation (200-600cGy). All received rabbit ATG (Thymoglobulin), initially at a total dose of 6mg/kg (2012-2013) and later at a total dose of 4.5mg/kg (2013 onward). Data was collected as part of study 01810588 registered at clinicaltrials.gov. Probabilities of relapse and non-relapse mortality (NRM) were generated using cumulative incidence estimates to accommodate competing risks. Probabilities of overall survival (OS) and progression-free survival (PFS) were calculated using Kaplan-Meier estimates. Results Our study included 220 haplo-cord transplant recipients with a median age of 58 yrs (Range: 18 – 76 yrs). The majority had myeloid disease (AML – 54%, MDS/MPN – 19.5%, CML – 2%). Conditioning incorporated TBI for 120 patients (55%). ATG was dosed at 6mg/kg for 41 patients (19%). The median absolute lymphocyte count (ALC) at first ATG exposure was 0.4 × 10^9/L (IQR25-75: 0.2-0.7 × 10^9/L). The median patient weight was 75.5kg (IQR25-75: 65.4-89.9kg). Neutrophil and platelet engraftment occurred at a median of 12 days (IQR25-75: 10-16 days) and 22 days (IQR25-75: 17-35 days) respectively. Graft failure was observed in 16 patients (7%). Rapid early recovery of CD19+ lymphocytes, CD56+ natural killer cells and IgG levels was observed. T-cell recovery was delayed, with a gradual increase observed from Day 100 (Figure). Overall PFS and OS at 2 yrs were 30% (CI 24-37%) and 39% (CI 32-46%) respectively. The 2 yr cumulative incidence of NRM was 33% (+/- 3% SEM). The overall 2 yr incidence of relapse was 37% (+/- 3% SEM), however there was a significant difference in relapse incidence between patients treated with 6mg/kg ATG (54%) versus 4.5mg/kg (31.5%) (p=0.009). Conclusion Haplo-cord transplant facilitates early neutrophil engraftment with early normalization of B- and NK-lymphocytes. An observed delay in T-cell recovery may be due to the use of ATG in this platform and higher ATG doses result in a greater incidence of relapse. Our data supports the minimization or replacement of ATG in haplo-cord transplant. An analysis of GVHD incidence and viral reactivation events is ongoing and will be presented.

Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

AimMesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs. Main MethodsWe used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers. Key FindingsCHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021–treated MSCs showed increased nuclear localization of β-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021–treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021–treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021–treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection. SignificanceMSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

Післязабійний кістковий мозок великої рогатої худоби – перспективне джерело стовбурових клітин

The development of modern methods of cryopreservation of animal cells has promoted to introduce the concept of creating of stem cell banks for their future use, to avoid problems with donor shortages. Large-scale cryopreservation of stem cells began in the 1990s with the establishment of human cord blood banks. The issue of long-term storage of stem cells is also acute in veterinary medicine. To obtain of cellular material, is needed a source suitable for cell isolation. As such a source it is advisable to consider a slaughter material from productive animals. The purpose of this study is to establish the possibility of cattle bone marrow use after slaughter as source of stem cells, on the basis of the proliferation index and the viability of the cultured cells to determine the suitability of this biological material for the obtaining of stem cells 72 hours after slaughter of the animal. Materials and methods. The bone marrow was obtained from the femur of a cow, 3 years old, who was slaughtered in a slaughterhouse. The bone marrow was collected with sterile tweezer into a sterile tube, filled with 0.25% trypsin solution (ratio of bone marrow volume to trypsin solution – 10: 1) and placed in a refrigerator (t + 4 0C) for 24 hours for enzymatic disaggregation. After bone marrow disaggregation, cultivation of cells was performed in a CO2-incubator using of disposable plastic Petri dishes (d = 30 mm) according to the standard procedure by periodically passaging them after forming a monolayer by 95–100%. Results. Cultivation of a suspension of cells, derived from the post-mortemal bone marrow of a cow, revealed that colonies of cells began to emerge 6–7 days after sowing. Passaging the cells with 0,25% trypsin-Versen solution and sowing them in new cultural plates contributed to increase the mass of cells that actively proliferated. It was established that stem cells, isolated from the post-mortemal bone marrow of cattle, have significant proliferative potential, as evidenced by proliferation indices  from I to III passages and high viability of cells. Thus, the post-mortemal bone marrow of the cattle can be used as an alternative source of stem cells. This biological material is suitable for the isolation of stem cells, even 72 hours after slaughtering the animal, which opens the prospects of its transportation over long distances.

Tissues from Post-Mortem Donors as Alternative Sources of Stem Cells for Regenerative Medicine.

Stem cells provide for all of the tissues in our body during embryogenesis. In adult organisms, they can be found as rare populations of tissue-specific stem cells in quiescent states, although they can still regenerate damaged tissues. Astonishingly, these cells are retained in tissues even post-mortem. There have been several reports that have provided evidence that cells with stem-like capabilities can be isolated, expanded, and differentiated in vitro from various tissues several hours, or even several days, post-mortem. Moreover, some post-mortem-tissue-derived stem cells can successfully engraft and regenerate injured host tissues. Here, we review in-vitro and in-vivo studies that provide evidence of isolation and characterization of stem cells from different tissues post-mortem, with a focus on the musculoskeletal and neural systems. Finally, we discuss their potential for use in regenerative medicine, and what needs to be done in further research toward their better exploitation.

Factors Associated With Umbilical Cord Blood Collection Quality in Japan.

BackgroundUmbilical cord blood (UCB) has become an established alternative source of hematopoietic stem cells with marrow and postmobilization peripheral blood. The presence of a large amount of clots may lead to the deterioration of cord blood quality. To improve UCB quality as a source of hematopoietic stem cells in Japan, we examined factors associated with UCB collection methods from the viewpoint of eliminating the presence of clots.MethodsIn August 2019, we requested the directors of 74 certified facilities to provide information on UCB collection methods in Japan. A total of 46 (62.2%) of them responded with valid information on a total of 2,892 UCB collections. In this study, collected UCB without clots macroscopically was evaluated as a high-quality UCB.ResultsThe 2,891 UCB collections described during the study period were divided to those with (n = 760, 26.3%) and without clots (high quality; n = 2,131, 73.7%). Multivariate analysis revealed single puncture as a factor determining high-quality UCB collection (adjusted odds ratio (ORs): 1.80, 95% confidence interval (CI): 1.3 - 5.4, P = 0.01).ConclusionsSingle puncture is an independent effective factor determining high-quality manual UCB collection in Japan.

Results of Unrelated Donor Hematopoietic Stem Cell Transplantation for Sickle Cell Disease in Europe on Behalf of Paediatric Diseases (PDWP) and Inborn Errors Working Parties (IEWP) of the EBMT

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) is, to date, the only curative treatment for sickle cell disease (SCD). Because a human leukocyte antigen (HLA) matched sibling donor is not always available, alternative stem cell sources such as unrelated or haploidentical related donors have been explored. The likelihood of finding a 10/10 (HLA-A, B, C, DRB1 and DQB1) matched donor varies among ethnic groups, with the lowest probability among individuals of African descent. To date, few series of SCD patients transplanted with an unrelated donor (UD) have been reported, but the high rates of rejection and chronic graft versus host disease (cGvHD) have limited its widespread application. Patients and methods: We report the results of a retrospective, registry based, survey on 70 UD HSCT performed in patients (pts) with SCD from UD in 22 European Society for Blood and Marrow Transplantation (EBMT) centers between 2005 and 2017. Data were collected from the EBMT database and missing information was updated by the centers. Median follow up was 38 (range 2-154) months. Most pts were HbSS (n=54; 78%), had positive serology for CMV (80%), and a Karnofsky score >80% (98%). Eighteen pts had a major ABO incompatibility. Recurrent vaso-occlusive crisis (n=58), cerebral vasculopathy (n=23) and acute chest syndrome (n=24) were the main indications for HSCT. Red blood cell (RBC) transfusions pre-HSCT were reported in 97% of pts of whom 53% received more than 20 transfusions; 14% of the transfused pts had RBC alloantibodies. Hydroxyurea pre-HSCT was used in 65% of pts. Median age at HSCT was 9.6 years (range 2-43) with 87% of pts being ≤ 16 years. Stem cell source was bone marrow (BM) in 55 pts (79%) and peripheral blood (PBSC) in 15 (21%). The median number of infused TNC /kg was 3.6 x 108 for BM and 7.1 x 108 for PBSC; the median number of infused CD34/kg was 4.4 x 106 for BM and 8.3 x 106 for PBSC. HLA matching at high resolution typing was 10/10 (HLA-A, B, C, DRB1 and DQB1) in 31, 9/10 in 17 and 8/10 in 4 of the patient-donor pairs; intermediate resolution typing was available for 10 (10/10 or 9/10) and the HLA information was missing for the remaining 8 patient-donor pairs. The most frequent conditioning regimens were fludarabine-thiotepa-treosulfan (64%) and busulfan- cyclophosphamide (12%). GvHD prophylaxis was cyclosporine plus methotrexate in 59%. Anti-thymocyte globulin was used in 90% and alemtuzumab in 9% of pts. Results: The cumulative incidence (CI) of neutrophil engraftment at 60 days was 93% (95% CI 76-100), with median time to engraftment of 18 days; platelet engraftment at 180 days was 90% (95% CI 83-98) with a median time of 20 days. Ten pts had graft failure (5 primary and 5 secondary) of whom 6 had a second transplant and were all alive at last FU (median 9.5 months after second HSCT). The CI of grade II-IV aGVHD at 100 days was 23% (95% CI 15-36), and 8 pts (11%) had grade III-IV. Acute GVHD was more frequent in patients who received PBSC (PBSC 42.9%, BM 18.2%, p=0.062). Three-year CI of cGVHD was 23% (95% CI 15-36), 7 pts (10%) had limited and 9 (13%) extensive cGvHD. Three-year overall survival (OS) was 90±4%; three-year event free survival (EFS) (considering death and graft failure as events) was 76±6%; HLA matching between donor and recipient was the most important factor for OS and EFS. Considering only pts-donor pairs with high resolution HLA typing available (n=52), 3-year OS was 96±4% in 10/10 group compared to 77±11% in 9/10 plus 8/10 group (p 0.065), 3-year EFS was 85±7% vs 62±12% (p 0.040), respectively. No significant differences between the groups were observed in CI of neutrophil engraftment, aGVHD and cGVHD. Conclusion: UD HSCT is a valid option for SCD patients who lack an HLA-identical sibling donor. Nevertheless, efforts are still needed to improve outcomes after UD HSCT. Our results indicate that using a 10/10 HLA matched UD improves both OS and EFS compared to donors with 1 or more mismatches; so, when such a matched unrelated donor is not found, using an haplo relative or an unrelated cord blood as donor source should be evaluated. A prospective trial is in preparation to evaluate the use of haploidentical donors for HSCT in SCD (EudraCT number: 2018-002652-33). Disclosures No relevant conflicts of interest to declare.

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.

Haplo-Cord Transplantation with Anti-Thymocyte Globulin: Comparative Clinical Outcomes with or without Routine G-CSF Administration

Introduction: Haplo-cord transplantation - the co-infusion of a single umbilical cord graft with CD34-selected cells from a haplo-identical donor, offers an alternative stem cell source for patients lacking HLA-matched donors. The T-cell depleted haplo-graft acts as a myeloid bridge until replaced by durable cord hematopoiesis. Anti-thymocyte globulin (ATG) is routinely used in haplo-cord preparative regimens to reduce graft failure, graft vs. host disease (GVHD) and graft vs. graft reactions. ATG depletes T-lymphocytes through Fas-mediated apoptosis, but also indirectly stimulates T-cell destruction through antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) (de Koning, Blood Adv. 2018). Excessive depletion of cord lymphocytes may compromise immune reconstitution and graft vs. tumor effects. G-CSF administration early post-transplant may sensitize cord lymphocytes to the cytotoxic effects of residual ATG. G-CSF drives myeloid precursor proliferation and activates phagocytosis, facilitating increased ADCP and ADCC of ATG-coated cells (de Koning, Blood Adv, 2018). Prior to the publication of this data, our institutional practice was to administer G-CSF to haplo-cord transplant recipients from day +6 until neutrophil engraftment. In April 2018, we altered our practice - withholding routine G-CSF treatment. We here compare our transplant outcomes 1 year before and after this practice shift. Methods: We examined consecutive adult patients with hematologic malignancy that underwent haplo-cord transplantation conditioned with fludarabine and melphalan with or without total body irradiation (400 cGy) at Weill Cornell Medicine, between 04/2017 and 04/2019. All patients received rabbit ATG (Thymoglobulin) 1.5 mg/kg on days -5, -3 and -1 (total dose 4.5 mg/kg). Data was collected as part of study 01810588 registered at clinicaltrials.gov. Probabilities of relapse, relapse-related mortality (RRM) and non-relapse mortality (NRM) were generated using cumulative incidence (CI) estimates to accommodate competing risks. Probabilities of overall survival (OS) and progression-free survival (PFS) were calculated using Kaplan-Meier estimates and inter-group comparison performed by log-rank testing. Variables with potential survival impact were evaluated in a Cox proportional hazards regression model. Results: The study included 59 haplo-cord recipients - 30 who received G-CSF (GCSF group) and 29 who did not (NO GCSF group). Groups were well matched for age, weight, disease, DRI, baseline lymphocyte count, CMV recipient/donor status and TBI treatment (Table). Neutrophil engraftment occurred at a median of 10 days in the GCSF group vs. 14 days in the NO GCSF group (p=0.0002), and platelet recovery at a median of 20 and 22 days (p=-0.61). Primary graft failure occurred in 1 GCSF patient and 2 NO GCSF patients (p=0.53). The incidence of acute GVHD (grade II-IV) by day 100 was 10% in both groups (p=0.97). There was no significant difference between GCSF and NO GCSF in the CIs of relapse, RRM or NRM at 1yr. No difference was found in 1yr OS or PFS on univariate analysis (Table). After adjusting for patient age, weight, lymphocyte count prior to ATG, disease subtype, CIBMTR disease risk index (DRI), Karnofsky performance status (KPS), comorbidity score and TBI exposure, in multivariate analysis, no effect on OS or PFS was observed. CMV reactivation was observed in 40% (GCSF) vs. 21% (NO GCSF) (p=0.11). The trend towards lower reactivations in the NO GCSF may in part be due to the introduction of letermovir prophylaxis for CMV positive transplant recipients in 02/2018. Acknowledging the limited follow up of NO GCSF patients, we note a trend towards improved CD4 counts at 1yr in this group - median 271 cells/μL (n=6, IQR25-75 = 240-342) vs. 155 cells/μL in the GCSF group (n=16, IQR25-75 = 111-473) (p=0.17). Conclusion: Our data shows that G-CSF can safely be eliminated from haplo-cord transplant. We see a delay in neutrophil engraftment but no adverse effect on early morbidity or mortality outcomes. We continue to withhold early G-CSF while assessing long-term outcomes. Longer follow-up is needed in our cohort to enable a systematic analysis of immune reconstitution. Early CD4+ cell recovery after ATG treatment has been shown to improve OS, PFS, NRM and RRM (Admiraal, Lancet Hem 2015 & 2017). Disclosures Van Besien: Miltenyi Biotec: Research Funding.

Pre-Engraftment Immune Reactions Has Unique Graft-Versus-Leukemia Effects after Single Cord Blood Transplantation

<Introduction> Cord blood transplantation (CBT) has now become one of the alternative stem cell sources for patients with a wide variety of hematological diseases. Considering that cord blood (CB) grafts contain T cells with an immunologically naïve phenotype, concerns have been raised regarding the capability of CB-derived lymphocytes to mediate sufficient graft-versus-leukemia (GVL) effects. However, the incidence of relapse after CBT is reportedly comparable to that after other stem cell sources. Moreover several recent studies have shown even lower relapse rate in CBT compared to the others, despite a lower incidence of GVHD. We and others have reported a unique clinical manifestation, termed pre-engraftment immune reactions (PIR), that develop early after CBT and before engraftment. The unique immune-mediated manifestation may have the potential to cause GVL effect from very early time point after CBT besides GVHD. <Methods> We conducted a retrospective study of 551 CB recipients to investigate the GVL effect of the PIR after CBT. Patients who underwent single CBT at our institute from 2004-2017 were included. Only those in non-remission status were included. Patients who had a prior history of allogeneic transplant, who had active infections, who had poor PS, and who received ATG or short-term MTX as GVHD prophylaxis were excluded. The diagnosis of PIR was made when patietns showed a high fever (=>38℃) and at least 2 of the following 5 symptoms (skin eruptions, diarrhea, jaundice, weight gain, and peripheral edema) developed six or more days before neutrophil engraftment. Patients who presented with severe organ damage, such as lung, kidney, or liver dysfunction, were classified as severe type. This study was approved by the institutional review board. <Results> The median age was 57 (range, 16-77) years. All patients had hematological malignancies and all were in non-remission status. Sixty-five percent of these patients received MAC regimens, 63% received tacrolimus plus MMF as GVHD prophylaxis, and 86% received a CB unit with 2 or 3 antigen-mismatch, and the median numbers of TNC and CD34-positive cells were 2.59x107/kg and 0.89x105/kg, respectively. The cumulative incidences of total PIR and severe PIR were 69.3% and 20.0%, respectively, with a median onset of 8 days (range, 5-19) post-CBT. Patients who developed PIR were treated with systemic corticosteroids at the discretion of the primary physicians. Younger age (<55 years) or a lower dose of CD34+ cells were identified as significant factors associated with the development of PIR on univariate analysis, and only younger age was significant on multivariate analysis. Patients with PIR (n=382) showed a significantly lower relapse rate than those without PIR (n=169) (27.2% vs 48.8% at 2 years, P<0.01), while there was no major difference in the non relapse mortality (NRM) rate between the two groups (35.7% vs 34.9% at 2 years, P=0.88) (Figure 1A and 1B). Patients with severe PIR (n=110) showed a more pronounced decrease in relapse incidence (12.5% in severe PIR group vs 33.1% in mild PIR group vs 48.8% in PIR (-) group, P<0.01). The cumulative incidence of NRM in the group, however, was extremely high compared to the others (73.3% in severe PIR group vs 20.8% in mild PIR group vs 34.9% in PIR (-) group, P<0.01). Patients with mild PIRs (n=272) showed reductions of both relapse and NRM compared to those without PIRs. The lower incidence of relapse in the PIR group was translated to better 2-year overall survival (OS) compared to those without PIR (41.2% in PIR (+) group vs 31.5% in PIR (-) group, P<0.01) (Figure 1C). Two-year OS of those with mild PIR was better than the other 2 groups (52.0% in mild PIR group vs 31.5% in PIR (-) group vs 13.9% in severe PIR group, P<0.01) (Figure 1D). Patients with severe PIR showed worse OS, mainly due to the higher incidence of NRM. A higher GVL effect was observed in patients with myeloid malignancies than in those with lymphoid malignancies. <Conclusions> Our data clearly showed that development of PIR after single CBT reduces relapse rate without increasing NRM and contributes to superior survival rate in advanced hematological malignanicies. PIR is one of the clinically important factors associated with unique GVL effects after single CBT. Figure 1 Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell transplantation and cellular therapy in sickle cell disease: where are we now?

Sickle cell disease (SCD) is a common monogenic disorder that is characterized by an A to T substitution in the β-globin gene that leads to the production of hemoglobin S (HbS). Polymerization of HbS leads to significant morbidity including vaso-occlusion, pain, hemolytic anemia, and end organ damage. Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment; however, suitable donors are not always readily available. This study reviews the current status of allo-HCT and autologous cellular therapies for SCD. Alternative sources of allogeneic stem cells from unmatched donors such as cord blood and haploidentical donors are gaining traction. Early experience has shown that better conditioning regimens and graft-versus-host disease prophylaxis are needed before these donor sources can gain widespread use. Clinical trials are underway to determine the feasibility and efficacy of autologous transplantation with gene modified hematopoietic stem cells. Gene therapy strategies include HbS gene correction, gene addition, and hemoglobin F induction. Preliminary results are very encouraging. Matched sibling allo-HCT for patients with SCD results in more than 90% overall survival and more than 80% event-free survival. Because only 25-30% of patients have a matched sibling donor, alternative donor options such as matched unrelated donors, related haploidentical donors and unrelated umbilical cord blood donors are being considered. Clinical trials investigating various strategies for gene therapy followed by autologous transplantation are underway. One major challenge is obtaining sufficient hematopoietic stem cells for gene therapy. Studies are being conducted on the optimal mobilization regimen and collection strategy.

Influence of alternative donor type on early survival after hematopoietic stem cell transplantation for acute myeloid leukemia lacking a sibling donor.

Allogeneic hematopoietic stem cell transplantation is the only potentially curative therapy for acute myeloid leukemia. In the absence of an HLA-matched related or unrelated donor (MRD or MUD), the best alternative donor source remains controversial. Umbilical cord blood and haploidentical donors offer a shorter delay from indication to transplantation. This retrospective multicentre study of a French registry compares overall survival in the 18 months following registration in the absence of a MRD between four types of donors. Between 2012 and 2016, 1302 patients were transplanted using MUD (control, n = 803), mismatched MUD (n = 219), umbilical cord blood (n = 153) and haploidentical (n = 127) donors. Multivariate analyses were conducted for overall survival after registration, after transplant, and transplant-related mortality. After adjustment for variables, the type of donor did not influence any of the three end points. Our results confirmed the significant negative impact of longer time between registration and transplant: HR = 1.04 [1.02-1.06] (p < 0.0001). This indicates a positive correlation between better survival and shorter registration-to-transplantation wait time. In the absence of a sibling donor, the alternative stem cell source does not impact early survival in acute myeloid leukemia patients. The minimization of registration-to-transplantation time should be considered when weighing the alternative donor options.