Cord Blood Stem Research Articles

EGR1 Haploinsufficiency Confers a Fitness Advantage to Hematopoietic Stem Cells Following Chemotherapy

Therapy-related myeloid neoplasms (t-MNs) share many clinical and molecular characteristics with AML de novo in the elderly. One common factor is that they arise in the setting of chronic inflammation, likely because of advanced age or chemotherapy-induced senescence. Here, we examined the effect of haploinsufficient loss of the del(5q) tumor suppressor gene, EGR1, commonly deleted in high-risk MNs. In mice, under the exogenous stress of either serial transplant or successive doses of the alkylating agent N-ethyl-nitrosourea (ENU), Egr1-haploinsufficient hematopoietic stem cells (HSCs) exhibit a clonal advantage. Complete loss of EGR1 function is incompatible with transformation; mutations of EGR1 are rare and are not observed in the remaining allele in del(5q) patients, and complete knockout of Egr1 in mice leads to HSC exhaustion. Using chromatin immunoprecipitation sequencing (ChIP-seq), we identified EGR1 binding sites in human CD34+ cord blood-derived stem and progenitor cells (HSPCs) and found that EGR1 binds genes critical for stem cell differentiation, inflammatory signaling, and the DNA damage response. Notably, in the chromosome 5 sequences frequently deleted in patients, there is a significant enrichment of innate and inflammatory genes, which may confer a fitness advantage in an inflammatory environment. Short hairpin RNA (shRNA)-mediated silencing of EGR1 biases HSPCs toward a self-renewal transcriptional signature. In the absence of EGR1, HSPCs are characterized by upregulated MYC-driven proliferative signals, downregulated CDKN1A (p21), disrupted DNA damage response, and downregulated inflammation-adaptations anticipated to confer a relative fitness advantage for stem cells especially in an environment of chronic inflammation.

Rapid potency assessment of autologous peripheral blood stem cells by intracellular flow cytometry: the PBSC-IL-3-pSTAT5 assay

Background aimsThe current gold standard for stem cell product potency assessment, the colony-forming unit (CFU) assay, delivers results that are difficult to standardize and requires a substantial amount of time (up to 14 days) for cellular growth. Recently, the authors developed a rapid (<24 h) flow cytometry assay based on the measurement of intracellular phosphorylated STAT5 (pSTAT5) in CD34+ cord blood stem and progenitor cells in response to IL-3 stimulation. The present work presents a novel adaptation of the protocol for use with autologous peripheral blood stem cells (PBSCs) and a performance comparison with the CFU assay. MethodsThe flow cytometry intracellular staining assay was optimized for PBSCs, and patient samples were analyzed using the PBSC-IL-3-pSTAT5 and CFU assays. Warming events were also simulated to emulate impaired potency products. ResultsOptimization led to minor protocol adjustments, such as removal of the red blood cell lysis step, the addition of a formaldehyde fixation step and an increase in anticoagulant concentration. The PBSC-IL-3-pSTAT5 assay discriminated between normal and impaired samples and identified 100% (18 of 18) of the impaired samples, thus showing better specificity than the CFU assay. ConclusionsThe updated IL-3-pSTAT5 potency assay has several important advantages, such as accelerating the release of autologous stem cell products and enabling the detection of potentially impaired products. The assay could also be used to rapidly assess the potency of any cryopreserved allogeneic stem cell product, such as those processed during the coronavirus disease 2019 pandemic.

Study of genetic variability of umbilical cord blood using RAPD assay

Background: The genetic variability of Umbilical Cord Blood (UCB) is most important for newborn screening, therapeutic possibility of haematological disorders as well as for the establishment of cord blood banking and stem cell research.&#x0D; Method: Genetic variability of umbilical cord blood (UCB) of 22 human subjects was evaluated first time by applying Random Amplified Polymorphic DNA (RAPD) assay using six decamar primers (B-14, OPB-05, OPB-08, OPB-12, OPB-19 and UBC-122).&#x0D; Result: A total number of bands were recorded 312 from 116 polymorphic loci and single monomorphic locus. All the markers showed highest polymorphism (100%) except the primer OPB 08 (92.31%) among tested individuals. The genetic distance was observed with highest 1.0 and lowest 0.72 respectively whereas mean genetic distance was recorded 0.90. Considering Shannon-Wiener index average diversity was recorded 0.139365. The mean Nei genetic similarity was found 0.17 which was found opposition to genetic distances. A phylogenetic relationship among the individual subjects was also observed between the linkage distances of 11 to 27 with 8 clades, 3 subclusters and a cluster. In addition, average allele frequency p and q was observed 0.08156 and 0.948751 respectively whereas highest intra locus gene diversity and average gene diversity were found 3.323817 and 0.144572 respectively.&#x0D; Conclusion: Considering different parameters, higher genetic variability was found among the experimental subjects, probably due to the mixture DNA of parents and newborn.&#x0D; Bangladesh Journal of Medical Science Vol.20(4) 2021 p.848-854

Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

830\xa0nm photobiomodulation therapy promotes engraftment of human umbilical cord blood-derived hematopoietic stem cells

Human umbilical cord blood (hUCB)-derived hematopoietic stem cells (HSCs) are an important source for HSCs in allogeneic HSC transplantation, but a limited number and a low efficacy of engraftment greatly restrict their clinical use. Here, we report the ability of photobiomodulation therapy (PBMT) to significantly enhance the engraftment efficacy of hUCB HSCs and progenitor cells (HSPCs). hUCB CD34+ cells were illuminated at a fluence of 2 J/cm2 with a near-infrared light (830 nm) transmitted by an array of light-emitting diodes (LED) prior to infusion of NOD/SCID-IL2Rγ−/− mice. The pre-treatment resulted in a threefold higher of the mean percentage of human CD45+ cells in the periphery of the mice compared to sham-treated CD34+ cells. The enhanced engraftment may result from a PBMT-mediated increase of intracellular reactive oxygen species (ROS) levels and Src protein phosphorylation in CD34+ cells. The two events were causally related as suggested by the finding that elevation of ROS by hydrogen peroxide increased Src phosphorylation, while ROS reduction by N-acetyl cysteine partially reversed the phosphorylation. The investigation demonstrates that PBMT can promote engraftment of hUCB HPSCs, at least in part, via ROS-mediated Src signaling pathway. PBMT can be potentially a safe, convenient, and cost-effective modality to improve hematological reconstitution in patients.

Screening for pre-leukemia TEL-AML1 chromosomal translocation in banked cord blood units: cord blood bank perspective.

Acute lymphocytic leukemia is the most common leukemia in children. Many studies suggest the existence of two subsequent hits in order for the disease to occur. TEL-AML1 (ETV6-RUNX1) is considered an initial genetic hit that occurs prenatally and generates a pre-leukemia clone. In cord blood (CB) stem cell transplantation, donor cell leukemia (DCL) is one of the complications associated with the presence of the pre-leukemic clone. The aim of this study was to identify the prevalence of ETV6-RUNX1 translocation in CB units and the feasibility in implementing such a screening test, to ensure the safety of the CB units. A total of 424 CB samples were tested from the CB units banked at KAIMRC-CBB. RNA was extracted and cDNA synthesis was performed on 1ug input RNA using Reverse Transcriptase RT-PCR methodology. Chromosomal translocation ETV6-RUNX1 was tested using real time quantitative PCR methodology. Our study showed undetectable levels of ETV6-RUNX1 in all tested CB samples. The samples were analyzed for the chromosomal translocation ETV6-RUNX1 under controlled conditions, using control and fusion genes with known concentrations. The result of this study does not rule out the importance of this screening test in predicting and/or preventing DCL. Moreover, the outcome strengthens the adopted system in our CBB for mother medical history screening prior to donation. We propose adding this test during the verification testing stage, prior to the release of CB units selected for transplantation rather than at the banking stage.

2020 – ACTIVATION OF THE RECEPTOR TYROSINE KINASE (RET) BY GDNF/GFRa1 IMPROVES CORD BLOOD-DERIVED HSC IN VITRO EXPANSION AND IN VIVO ENGRAFTMENT

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRa1), significantly improves the outgrowth of HSCs in vitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (∼1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (∼1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRa1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRa1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRa1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRa1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRa1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFkB activation and reduced reactive oxygen species, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRa1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation.

2019 – ASYMMETRIC CELL DIVISION MODULATES HUMAN HEMATOPOIETIC STEM CELL FATES

Effective manipulation of clinically relevant umbilical cord blood-derived human hematopoietic stem cell (hHSC) fate decisions remains an important goal for improved therapies. Based on observations in other model organisms, it was postulated that asymmetric cell division (ACD) regulates hHSC fate decisions of self-renewal and differentiation. In ACD, daughter cell fates are determined during cell division, e.g., by the asymmetric inheritance of cell fate determinants. Previous studies suggested the asymmetric inheritance of factors during progenitor divisions. However, due to the lack of tools for sufficient prospective hHSC enrichment, quantification of asymmetric inheritance and future daughter cell behaviors in the same experiment, hHSC ACD remained hypothetical. Here, we use continuous quantitative long-term single-cell imaging of highly purified hHSCs to identify factors and organelles that are asymmetrically inherited during hHSC divisions. Importantly, this asymmetric inheritance correlates with future asymmetric adhesion, motility, cell cycle progression, marker production, and metabolic activity in hHSC daughters. We further show, using novel real-time computational differentiation landscapes and in-silico hHSC purification, that this asymmetric inheritance also correlates with future expression of stem cell markers, and that culture conditions known to expand hHSCs increase symmetric cell divisions. We thus provide the first direct quantitative evidence that ACD exists in human hematopoietic stem and progenitor cells and modulates future daughter cells fates by the inheritance of factors during division. We provide insights into the underlying molecular mechanisms, and the exciting possibility to manipulate ACD and hHSC fate decision for improved therapy.

Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.

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.

Human cord blood-derived primitive CD34-negative hematopoietic stem cells (HSCs) are myeloid-biased long-term HSCs residing at the apex of human HSC hierarchy

Human cord blood-derived primitive CD34-negative hematopoietic stem cells (HSCs) are myeloid-biased long-term HSCs residing at the apex of human HSC hierarchy

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

Transplantation of human dopaminergic neurons differentiated from umbilical cord blood-derived induced pluripotent stem cells into the rat striatum

Transplantation of human dopaminergic neurons differentiated from umbilical cord blood-derived induced pluripotent stem cells into the rat striatum

Comparison of PPAR? DNA Methylation in Cord Blood and Human Mesenchymal Stem Cells to Assess Efficacy of Blood as Proxy Tissue for Obesity Studies

Comparison of PPAR? DNA Methylation in Cord Blood and Human Mesenchymal Stem Cells to Assess Efficacy of Blood as Proxy Tissue for Obesity Studies

Abstract 375: A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells

Abstract Introduction The adenosine deaminase acting on RNA (ADAR) family of RNA editases has been linked to the pathogenesis of diverse malignancies, including leukemia, breast cancer and hepatocellular carcinoma. We previously showed that human leukemia stem cells (LSC) from blast crisis (BC) chronic myeloid leukemia (CML) patients harbor increased ADAR1 expression compared with normal and chronic phase (CP) progenitors. Whole transcriptome RNA sequencing (RNA-Seq) revealed increased adenosine to inosine (A-to-I) RNA editing during CML progression concentrated within primate specific Alu-containing transcripts. However, detection of RNA editing by RNA-Seq in rare cell populations can be technically challenging, costly and requires PCR validation. Thus, the objectives of this study were to validate RNA editing of a subset of these LSC-associated transcripts in the context of lentivirally enforced ADAR1 expression, and to develop an RNA editing reporter reporter assay in human leukemia cells and a qPCR-based diagnostic test to rapidly detect CSC-associated aberrant RNA editing. Methods The BCR-ABL+ human leukemia cell line K562 was stably transduced with lentiviral human ADAR1 or vector. FACS-purified K562-ADAR1 cells were transfected with a luciferase-based reporter vector to confirm RNA editing activity. Two genes, MDM2 and APOBEC3D, were selected from our previous RNA-Seq studies of BC progenitors (Jiang et al, 2013). Targeted sequencing was performed on high fidelity PCR products using primers flanking each of 2 editing sites in each gene. RNA editing-specific qPCR primers were designed for each editing site using an allele-specific strategy that detects cDNA containing either an A or G(I) representing an RNA editing event. Both targeted sequencing and qPCR were used to detect RNA editing in K562-ADAR1 and primary cord blood-derived hematopoietic stem cells (HSC) lentivirally transduced with ADAR1. Results Lentivirally enforced ADAR1 expression promoted RNA editing activity as measured by luciferase reporter activity. Increased A-to-I changes in MDM2 and APOBEC3D were confirmed by targeted sequencing. In independent experiments, RNA editing site-specific qRT-PCR accurately detected RNA editing in K562-ADAR1 cells (n=3) and in primary HSC overexpressing ADAR1 (n=4). Site-specific primers distinguished G(I) bases at RNA editing sites in cDNA and as predicted gave no signal in gDNA. Relative A-to-I RNA editing ratios were increased by 2 to 3 fold in ADAR1-expressing cells at all four sites. Conclusions These results set the stage for development of primate-specific RNA editing as a novel diagnostic strategy for clinical LSC detection and identify ADAR1 as a potential therapeutic target in LSC. These data shed new light on the mechanisms of ADAR1-mediated generation of malignant progenitors that drive therapeutic resistance, disease progression and relapse in CML and may be applicable to other CSC-driven malignancies. Citation Format: Leslie A. Crews, Qingfei Jiang, Maria A. Zipeto, Angela C. Court, Christian L. Barrett, Marco A. Marra, Kelly A. Frazer, Catriona H. M. Jamieson. A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 375. doi:10.1158/1538-7445.AM2014-375

Timing of cord clamping in very preterm infants: more evidence is needed

In December 2012, the American College of Obstetricians and Gynecologists published a Committee Opinion entitled "Timing of umbilical cord clamping after birth." It stated that "evidence exists to support delayed cord clamping in preterm infants, when feasible. The single most important benefit for preterm infants is the possibility for a nearly 50% reduction in IVH." However, the Committee Opinion added that the ideal timing of umbilical cord clamping has yet to be determined and recommended that large clinical trials be conducted in the most preterm infants. Published randomized controlled trials include <200 infants of <30 weeks' gestation, with assessments of neurodevelopmental outcome in less than one-half of the children. This is a major gap in the evidence. Without reliable data from randomized controlled trials that optimally include childhood follow-up evaluations, we will not know whether delayed cord clamping may do more overall harm than good. Ongoing trials of delayed cord clamping plan to report childhood outcomes in >2000 additional very preterm infants. Current recommendations may need to change when these results become available. Greater international collaboration could accelerate resolution of whether this promising intervention will improve disability-free survival in about 1 million infants who will be born very preterm globally each year.

Drug a enhances the immunosuppressive properties of human umbilical cord blood-derived mecenchymal stem cells

Drug a enhances the immunosuppressive properties of human umbilical cord blood-derived mecenchymal stem cells

Haploidentical Peripheral Blood Stem Cell Transplantation Using Non-Myeloablative Conditioning With Post-Transplant Cyclophosphamide Regimen Is Safe and Is Associated With Very Encouraging Post-Transplant Outcomes

Introduction The availability of HLA matched donors remains a major obstacle for successful allogeneic hematotopoietic cell transplantation. The use of HLA-mismatched alternate donors such as cord blood and haploidentical donor stem cell sources have allowed for greater access for those patients who need an allo-HSCT but lack a suitable matched sibling or unrelated donor. Introduction of high dose cytoxan in the early post-transplant period has significantly improved the outcomes of patients undergoing haploidentical transplantation and has eliminated the need for expensive and labor-intensive ex-vivo T cell depletion. Encouraging results have been reported using this platform with bone marrow as the source of stem cells. However, there have been only limited reports using this transplant platform with G-CSF mobilized peripheral blood stem cells (PBSC) as a source of stem cells for haloidentical transplantation. Here we report the outcomes of 18 patients who underwent haploidentical transplant for hematological malignancies from single institution treated on the Hopkins non-meloablative conditioning regimen but with G-CSF mobilized PBSC as a source of stem cells from a haplo-identical family donor. Patients and Methods A total of 18 patients (median age 41 years, range 22-73 years, 11 males and 7 females) between July 2009 and June 2013 underwent haploidentical transplant at Washington University School of Medicine in St Louis using the Hopkins non-myeloablative conditioning regimen with post transplant cytoxan (fludarabine (30 mg/m2/day on days -6 to -2), cytoxan (14.5 mg/kg/day on days -6 and -5) and TBI (single dose at 200cGy on day -1) and all these patients received two doses of post-transplant cytoxan (50mg/kg on D+3 and D+4). G-CSF mobilized PBSC from parents (n=9) or siblings (9) were used as a graft source with median CD34+ cell dose of 5.0 x 106/kg and median CD3+ T cell dose of 19.7 x 107/kg. GVHD prophylaxis regimen included MMF plus tacrolimus (16/18 patients) or MTX plus tacrolimus (2/18 patients). Median follow-up of all patients was 251 (range 17-1174) days. Diagnoses included AML (n=12), ALL (n=2), NHL (n=2), CLL (n=1) and aplastic anemia (n=1). 7 out of 12 AML patients underwent transplant with active disease (not in remission) and 4/18 of these patients had prior history of allogeneic HCT. Results 16 patients (89%) engrafted (&gt; 95% donor chimerism), median time to neutrophil engraftment was 15 days (range: 12-28 days) and median time to platelet engraftment was 18 days (range: 11-40 days). None of these patients had secondary graft failure. 1-year overall survival (OS) for all patients was 62% and 100-day and 1-year non-relapse mortality (NRM) rates were 11% and 17% respectively. Both 1-year and 2-year relapse free survival (RFS) rates were 53%. Despite very high CD3+ T cell doses, cumulative incidence of grade II-IV aGVHD was 40.7% while grade III-IV aGvHD occurred in only 3 patients (17%). Cumulative incidence of cGVHD at 1 and 2 years were both at 8% (extensive in only 1 patient). CMV reactivation occurred in 11 patients (61%) but did not significantly impact their survival or relapse rates and none of these patients developed CMV disease. Conclusions Here we report the outcomes of 18 patients with hematologic malignancies or marrow failure states undergoing haploidentical transplant using the published Hopkins NMA conditioning platform with post-transplant high dose cytoxan and with G-CSF mobilized PBSC as a source of donor stem cells. In spite of the limited numbers of patients transplanted, our results suggest that this approach is both safe and effective and associated with rapid multilineage engraftment, low rates of both aGvHD and cGvHD and encouraging overall and disease-free survival rates and low rates of NRM. Based on these results, 1) G-CSF mobilized PBSC from haploidentical donors should be considered as an alternative source of haploidentical stem cells to BM and 2) future randomized trials using this platform to test the role of haploidential G-CSF mobilized PBSC with other unrelated donor stem cell sources (cord blood and matched unrelated could also be considered in the future. Disclosures: Abboud: Alexion: Honoraria; Ariad: Honoraria; Novartis: Honoraria; Teva: Speakers Bureau.

Unrestricted somatic stem cells as vehicle for nerve growth factor gene transfer

Objective: Nerve growth factor (NGF), a member of the neurotrophic factor family, plays a critical role in the maintenance and regeneration of different types of neurons. To overcome drastic challenges in the peripheral delivery of NGF, transplantation of NGF secreting stem cells to the target site of an injury may be an effective procedure. Unrestricted somatic stem cells (USSCs), a subtype of umbilical cord blood (UCB) stem cells, have shown promise for gene therapy purposes, and proper results have been observed from transplantation experiments in neurodegenerative disorders. Based on the considerable potential of USSCs for gene delivery applications, the goal of the current study was to establish a betaNGF gene containing USSCs, which is able to secrete functional recombinant betaNGF protein.Methods: Unrestricted somatic stem cells were isolated from UCB and were cultured in a DMEM medium. The betaNGF gene was cloned in the EFalpha-promoting lentiviral vector, and virus production was performed as a third generation lentivirus packaging system. Enzyme-linked immunosorbent assay, real-time PCR, co-culture, MTT, and immunocytochemistry assays were performed to evaluate the genetically engineered USSCs.Results: Overexpression of betaNGF gene in human USSCs created a USSC line that is able to secrete high amounts of functional betaNGF protein. betaNGF-secreting USSCs showed a high rate of viability along with acceptable immunological and morphological properties for transplantation into the nervous system.Discussion: Long-term expression of functional betaNGF, high viability of betaNGF producing USSCs, and expression of primary neuronal markers suggest that USSCs may be useful to deliver betaNGF into targeted sites of the nervous system in neurodegenerative disorders.