Infusion of a Cryopreservable Human Megakaryocyte-Biased Cell Product Results in Sustained Platelet Reconstitution In Vivo

Abstract The demand for platelet (PTL) transfusions has steadily increased, straining a supply that is limited by its dependency on donors, short lifespan, risk of infections and alloimmunization. This stimulated the search for alternative PTL sources including PTLs generated ex vivo from primary CD34+ cells and immortalized pluripotent stem cells. These approaches, however, are associated with obstacles such as scalability and encounter identical limitations as donor PTLs: short shelf life, storage at ambient temperature and limited lifespan after infusion. These obstacles lead us to focus our efforts on not producing PLTs but rather a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTLs after transfusion into patients. Umbilical cord blood units (CBU) are readily available sources for stem cell for transplantation. We created an efficient and cost effective culture system in which CB-derived CD34+ cells are first expanded then allowed to mature into MKs. Initially, we determined the optimal culture period (10-11 days) resulting in the greatest number of CD41+/CD42b- and CD41+/CD42b+ MKs which are capable of PTL production. Next, we used research and clinical grade CBU to generate clinically relevant doses of MK. The median number of CD34+ cells selected from one CBU was 2.5x106 with a purity of 90% (n=4). Following expansion and MK maturation, these cells generated 5.8x107 viable total nucleated cells (TNC)/CBU. Out of these, 3.3 x 107 were CD41+ MKs which corresponds to a median cell dose of 4.1x105 CD41+ cells/Kg of body weight. 92% of CD41+ MKs were mature CD42b+ cells which we previously showed that are capable of ex vivo platelet production. Finally, we performed clonogenic assays and found that one CBU can generate ~1.5x106 CFU-MK. One half of these MK-biased cultures was characterized and assessed immediately after culture and the other half was cryopreserved. The fresh product was infused into sublethally irradiated NSG mice and the presence of human PTLs in the mouse peripheral blood (mPB) was evaluated weekly for 8 weeks at which time the animals were also analyzed for hMK chimerism in the bone marrow (BM), spleen (SP) and lung. The results demonstrate that 87% of animals (13 out of 15) had a robust hPTLs population in their PB. hPTL were detected as early as week 1 post infusion and their number peaked on week 4 (median, 6x103 hPTL/μl) after which it plateaued. The release of hPTL in the mPB was accompanied by the presence of hCD41+ MKs in the mBM, SP and the lung indicating that the infused cells provided both early hPTL release and a reservoir of MK precursors for continuous hPTL production. We also found that in addition to MKs, these same organs contained hCD34+, CD45+ and myeloid CD15+ cells. These findings underscore the capabilities of this product which might have broader clinical applicability such as utilization during myeloablative or suppressive chemo/radiotherapy to improve the time and duration for both neutrophil and platelet engraftment. Equally important, we provide novel evidence that the lung is a site for hMK engraftment after transplantation, in line with recent reports recognizing the pulmonary bed as site for platelet production in the mouse. The major advantage of developing a MK-based product over ex vivo generated PTLs is the amenability of the former to cryopreservation thus becoming a readily available cellular therapy which would be amenable to stock-piling. Therefore, portions of the same MK products described above were cryopreserved then subjected to ex vivo and in vivo studies identical to these performed on their fresh counterparts. Following thawing, the average recovery rate was 71% for TNC and 74.3% for CD41+ cells. MK phenotype and morphology as well as the number CFU-MK generated ex vivo were identical to that found in the fresh product. Although the number of TNC in the thawed product was lower than that of its fresh counterpart, the number of hPTL detected after its infusion ranged from 0.4 to 20.5x103 hPTL/μl which is comparable to that detected after infusion of the fresh equivalent, 0.7-16x103 hPTL/μl. In summary, we created a potent transfusable MK cell product that provides robust and sustained PTL and hematopoietic engraftment in vivo and maintains this capability after cryopreservation. Clinical development of such product is now being pursued for the treatment of thrombocytopenia in acute leukemia patients undergoing chemotherapy. Disclosures Hoffman: Summer Road: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding; Merus: Research Funding; Incyte: Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding.

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311 EX VIVO-EXPANDED PERIPHERAL CD34+ CELLS FOR CHRONICALLY INJURED LIVER TREATMENT: IN VITRO AND IN VIVO STUDIES

Journal of Hepatology ◽

10.1016/s0168-8278(13)60313-x ◽

2013 ◽

Vol 58 ◽

pp. S130-S131

Author(s):

T. Nakamura ◽

T. Torimura ◽

H. Masuda ◽

H. Iwamoto ◽

O. Hashimoto ◽

...

Keyword(s):

Ex Vivo ◽

In Vivo Studies ◽

Cd34 Cells ◽

Injured Liver

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Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽

10.1182/blood.v104.11.2844.2844 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2844-2844

Author(s):

Ian K. McNiece ◽

Jenny Harrington ◽

Joshua Kellner ◽

Jennifer Turney ◽

Elizabeth J. Shpall

Keyword(s):

Cord Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

In Vivo Studies ◽

Adherent Cells ◽

Blood Products ◽

Cd34 Cells ◽

Duke University

Abstract Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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Dose Dependent Enhancement Of Neutrophil Recovery By Infusion Of Notch Ligand Ex Vivo Expanded Cord Blood Progenitors: Results Of a Multi-Center Phase I Trial

Blood ◽

10.1182/blood.v122.21.297.297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 297-297 ◽

Cited By ~ 4

Author(s):

Colleen Delaney ◽

Filippo Milano ◽

Ian Nicoud ◽

Shelly Heimfeld ◽

Chatchada Karanes ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Graft Failure ◽

Ex Vivo ◽

Chronic Gvhd ◽

Advisory Committees ◽

Neutrophil Recovery ◽

Cd34 Cells ◽

Primary Graft Failure

Abstract Introduction There is a strong clinical need to overcome the increased early non relapse mortality (NRM) associated with delayed neutrophil recovery following cord blood transplant (CBT). Therefore we established a methodology using Notch ligand (Delta1) as a strategy for increasing the absolute number of marrow repopulating CB hematopoietic stem/progenitor cells (HSPC). We previously reported preliminary results of the first 10 patients in 2010 demonstrating the ability of Notch-expanded CB HSPC to provide rapid myeloid recovery post-CBT.1 Herein we present the updated results on 23 patients accrued to this trial aimed at assessment of efficacy as well as the feasibility of overnight shipment of the expanded cell product to three outside institutions. Methods Between July 2006 and March 2013, 23 patients with hematologic malignancies were enrolled in this prospective multi-center Phase I trial coordinated by the Fred Hutchinson Cancer Research Center in which one CB unit was ex vivo expanded prior to infusion. Conditioning consisted of Fludarabine (75mg/m2), Cyclophosphamide (120mg/kg) and TBI (13.2 Gy) over 8 days. On day 0, the unmanipulated CB unit was infused first followed 4 hours later by infusion of the freshly harvested expanded CB cells. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. All CB grafts were 4-6/6 HLA-matched (A/B antigen level, DRB1 allele level) to the recipient. Engraftment, NRM, relapse and GVHD were calculated using cumulative incidence rates to accommodate competing risks. Overall survival was analyzed using Kaplan-Meier estimates. Results Patient diagnosis was AML (n=16), ALL (n=5) and biphenotypic leukemia (n=2). Nine patients (39%) were ≥CR2 and 5 were MRD+ at the time of transplant. Median age was 28 years (range, 4-43) and weight 70 kg (range, 16-91) with a median follow-up of 614 days (range, 271-2443). 22 patients received the expanded graft with one product not meeting release criteria. The cell doses infused were significantly higher in the expanded CB graft: 2.7 (1.5-6.3) vs 6.9 (0.4-27.6) x107 TNC/kg, p<0.0008; 0.15 (0.02-0.57) vs 7.7 (0.62-49.5) x106 CD34/kg, p<0.0001. HLA-matching and ABO incompatibility of the expanded and unmanipulated products were similar. The incidence of neutrophil recovery was 95% (95% CI, 71-100) at a median of 13 days (range, 6-41 days) among the 22 patients receiving expanded CB cells which is significantly faster than that observed in 40 recipients of two unmanipulated units otherwise treated identically at a median time of 25 days (range, 14 to 45; p<0.0001). The incidence of platelet recovery (>20 x 10^9/L) was 77% (CI 95%: 53- 89) by day 100 at a median of 38 days (range, 19 – 134). There was one case of primary graft failure. Importantly, rate of neutrophil recovery correlated with CD34+ cell dose/kg with 8 out of 11 patients receiving greater than 8x106 CD34+cells/kg achieved an ANC ≥ 500/µl within 10 days. 21 patients were evaluable for in vivo persistence of the expanded cells. Ten (48%) demonstrated in vivo persistence beyond one month post infusion. The expanded cell graft was persistent at day 180 in 7 patients, and in those that survived to one year, dominance of the expanded cell graft persisted in one patient. The incidences of grade II-IV and III-IV acute GVHD was 77% (95% CI, 53-89) and 18% (95% CI, 5-36%), respectively; mild chronic GVHD was observed in 4 patients and severe chronic GVHD in one. Probability of OS was 62% (95% CI, 37-79%) at 4 years. Notably, the cumulative incidence of NRM at day 100 was 8% (95% CI, 14-24%) and at 4 years was 32% (95% CI, 8-40%). Nine patients died at a median time of 216 days (range, 31-1578 days) with respiratory failure/infection the most common cause (n=6). There were two relapses at day 156 and 365 post-transplant, with one death due to relapse. Secondary malignancy and primary graft failure were the other 2 causes of death. Conclusions Infusion of Notch-expanded CB progenitors is safe and effective, significantly reducing the time to neutrophil recovery and risks of NRM during the first 100 days. An advantage for infusion of higher numbers of CD34+ cells/kg further demonstrates the need to develop methods that reproducibly provide even greater expansion of repopulating cells than currently achieved to improve efficacy and potentially cost effectiveness. 1. Delaney C, et al, Nat Med. 2010 Feb;16(2):232-6. Disclosures: Delaney: Novartis: DSMB, DSMB Other; Biolife: Membership on an entity’s Board of Directors or advisory committees; medac: Research Funding. Wagner:Novartis: Research Funding; cord use: Membership on an entity’s Board of Directors or advisory committees.

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T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽

10.1182/blood-2019-131491 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2673-2673

Author(s):

Jessica Liegel ◽

Dina Stroopinsky ◽

Haider Ghiasuddin ◽

Adam Morin ◽

Marzia Capelletti ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Cellular Therapy ◽

Ex Vivo ◽

Advisory Board ◽

Adoptive Cellular Therapy ◽

Advisory Committees ◽

Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

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Extracellular Tyrosyl-tRNA Synthetase Is a Potent Stimulator of Thrombocytopoiesis

Blood ◽

10.1182/blood.v128.22.1476.1476 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1476-1476

Author(s):

Sachiko Kanaji ◽

Taisuke Kanaji ◽

My-Nuong Vo ◽

Alessandro Zarpellon ◽

Ryan Shapiro ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Trna Synthetase ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Platelet Production ◽

Equity Ownership

Abstract Aminoacyl-tRNA synthetases (aaRSs) are enzymes with a key role in the first step of protein synthesis by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. During evolution, eukaryotic aaRSs have acquired additional domains and motifs conferring non-canonical functions beyond translation, such as expressing multiple cytokine activities. Repurposing aaRSs often requires an activation step and the first reported example was for human tyrosyl-tRNA synthetase (YRS), which is abundant in platelets and released from their α-granules upon thrombin or arachidonic acid stimulation. As shown by previous work, activated YRS (YRSACT) - created by natural proteolysis, alternative splicing, or rational mutagenesis - can express the activity of different cytokines. In the current study, we demonstrate that recombinant YRSACT rendered active by the gain-of-function mutation Tyr341Ala exhibits a previously unrecognized role in megakaryocytopoiesis and thrombocytopoiesis. When administered in vivo in C57BL/6 wild type (WT) mice, recombinant YRSACT caused platelet increase both under baseline conditions as well as in a model of immune-mediated thrombocytopenia in which mice are made thrombocytopenic by injection of rat anti-mouse glycoprotein (GP) Ib monoclonal IgG. When WT mouse bone marrow (BM) cells were cultured ex vivo for 3 days, YRSACT treatment increased the number of megakaryocytes by 3.0-fold, particularly of megakaryocytes with 16N ploidy. This effect was independent of thrombopoietin (TPO) signaling because YRSACT could support the expansion of c-mpl-/- (TPO receptor knock-out) mouse megakaryocytes. YRSACT had no effect on purified mouse CD41+ or Sca1+ hematopoietic progenitor cells, indicating that YRS-dependent stimulation likely required the contribution of other cells present in BM cultures. When mouse BM cells were stimulated with different doses of YRSACT, the number of F4/80+ monocyte/macrophages as well as of megakaryocytes increased in a dose-dependent manner. Mechanistic analysis revealed YRSACT targets the Toll-like receptor (TLR) pathway signaling through MyD88 in monocyte/macrophages, thereby enhancing release of cytokines that influence megakaryocyte development. In vitro binding assay showed that YRSACT is capable of binding to TLR2 and TLR4. The effect of YRSACT was attenuated in the BM cells derived from TLR2-/- mice and was abolished in MyD88-/- mice. Among the cytokines with synthesis induced by YRSACT, IL-6 plays a pivotal role in megakaryocyte development. Thus, we tested the effect of YRSACT on megakaryocytes obtained by culturing BM cell derived from IL-6-/- mice and found that no effect was apparent. The stimulatory effect of YRSACT on megakaryocytopoiesis was confirmed with human CD41+ megakaryocyte progenitors differentiated from CD34+ hematopoietic stem cells derived from peripheral blood. In conclusion, we have documented a previously unrecognized activity of YRSACT that results in enhanced megakaryocytopoiesis and platelet production. These studies document a mechanistically distinct aaRS-directed hematological activity that highlights new potential approaches to stimulating platelet production for treating thrombocytopenia and for improving ex vivo preparation of platelet concentrates for transfusion. Disclosures Belani: aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Do:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Yang:aTyr Pharma: Consultancy, Patents & Royalties, Research Funding. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v128.22.3859.3859 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3859-3859

Author(s):

Helen Fong ◽

Goar Mosoyan ◽

Ami Patel ◽

Ronald Hoffman ◽

Jay Tong ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Shelf Life ◽

Ex Vivo ◽

Culture Conditions ◽

Hematopoietic Stem ◽

Cell Product ◽

Cd34 Cells

Abstract Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients. Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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rAAV-based brain slice culture models of Alzheimer’s and Parkinson’s disease inclusion pathologies

Journal of Experimental Medicine ◽

10.1084/jem.20182184 ◽

2019 ◽

Vol 216 (3) ◽

pp. 539-555 ◽

Cited By ~ 9

Author(s):

Cara L. Croft ◽

Pedro E. Cruz ◽

Daniel H. Ryu ◽

Carolina Ceballos-Diaz ◽

Kevin H. Strang ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Transgene Expression ◽

Brain Slice ◽

Ex Vivo ◽

Three Dimensional ◽

Cost Effective ◽

In Vivo Studies ◽

Slice Culture

It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer’s and Parkinson’s disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC “toolkit” enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.

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Transient Inhibition of Transforming Growth Factor-β1 in Human Blood-Derived CD34+ Cells Enhances Survival and Proliferation in Vitro and Vascular Reparative functions In Vivo.

Blood ◽

10.1182/blood.v114.22.3546.3546 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3546-3546

Author(s):

Stephen Bartelmez ◽

Ashay Bhatwadekar ◽

Patrick Iversen ◽

Francis W Ruscetti ◽

Maria Grant

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Cxcr4 Expression ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership ◽

Tgf Β1

Abstract Abstract 3546 Poster Board III-483 CD34+ cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation as well as diminished migration prowess which is largely due to lower levels of bioavailable nitric oxide (NO). We asked whether a transient TGF-β1 blockade in CD34+ cells from diabetics would improve their reparative ability given that TGF-β is a key factor modulating stem cell quiescence. Peripheral blood lin-CD34+ cells or lin-CD34+CD38+/− cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1 -PMO), demonstrated to inhibit TGF-β1 protein expression in stem cells. Cells were then analyzed for cell surface TGF-β Receptor 2 (TGF-β R2) and CXCR4 expression, their ability to generate NO, their ability to migrate toward SDF-1, their ability to survive in the absence of added growth factors, and tested in vivo for their vascular reparative ability. After TGF-β1-PMO treatment, healthy and diabetic CD34+CD38+ and - cells downregulated TGF-βR2, upregulated CXCR4 expression, survived in the absence of added growth factors ex vivo and migrated more efficiently to SDF-1 compared to controls. TGF-β1-PMO treated diabetic CD34+ cells restored NO production to non-diabetic levels. In contrast, TGF-β1-PMO did not enhance NO generation in CD34+ cells from healthy subjects. Using an in vivo retinal ischemia reperfusion model, we observed that TGF-β1-PMO treatment increased the ability of both healthy and diabetic CD34+ cells to home to injured capillaries compared to control PMO treated cells. As also observed in our current study, a reduction of TGF-β1 levels in murine hematopoietic stem cells correlates with a reduction in TGF-βR2 expression which may induce proliferation in vivo. We also show that both diabetic and healthy lin-CD34+CD38+ cells express TGF-βR2 by FACS. In contrast, only healthy lin-CD34+CD38- cells expressTGF-βR2 while diabetic lin-CD34+CD38 - cells express essentially no cell surface TGF-βR2 (<5 % of cells are TGF-βR2+). Our results suggest that a transient blockade of TGF-β1 may represent a promising therapeutic strategy in restoring vascular reparative function in diabetic CD34+ cells. Disclosures: Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Head, SRB, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iversen:AVI-Biopharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Safety and Exploratory Efficacy Of Ex Vivo Expanded Umbilical Cord Blood (UCB) Hematopoietic Stem and Progenitor Cells (HSPC) Using Cytokines and Stem-Regenin 1 (SR1): Interim Results Of a Phase 1/2 Dose Escalation Clinical Study

Blood ◽

10.1182/blood.v122.21.698.698 ◽

2013 ◽

Vol 122 (21) ◽

pp. 698-698 ◽

Cited By ~ 6

Author(s):

John E. Wagner ◽

Claudio G Brunstein ◽

David McKenna ◽

Darin Sumstad ◽

Suzanne Maahs ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Phase 1 ◽

Median Number ◽

Hematopoietic Stem ◽

Hematopoietic Malignancy ◽

Neutrophil Recovery ◽

Increased Risk ◽

Cd34 Cells

Abstract Background Despite enhanced tolerability of HLA mismatch, the reduced number of HSPC in an UCB graft limits the use of this stem cell source because of delayed hematopoietic recovery and increased risk of graft failure, particularly in adults. For this reason, we explored the effectiveness of SR1, an aryl hydrocarbon receptor antagonist, in the presence of cytokines to expand HSPC ex vivo prior to transplantation. Patients and Methods Nine patients with high-risk lympho-hematopoietic malignancy and two partially HLA (4-6/6)-matched UCB units were treated with cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2 and total body irradiation 1320 cGy followed by double UCB transplantation. Unit1 (the unmanipulated larger of the two units) was infused followed by SR1-expanded UCB HSPC (derived from CD34 selected Unit2 cells cultured for 15 days) as well as rethawed CD34 negative Unit2 cells. GVHD prophylaxis was cyclosporine A and mycophenolate mofetil. Results Culture in the presence of SR1 resulted in a median of 248-fold (range, 66-446) expansion of CD34+ cells. The median number of CD34+ cells and CD3+ cells infused for Unit1 and Unit2 were 0.3 x 106/kg (range, 0.2-0.9) and 11.0 x 106/kg (range, 1.4-48.9), and 7.3 x 106/kg (range, 4.6-10.6) 2.8 x 106/kg (range, 0.4-4.9), respectively. There were no infusional toxicities noted. Based on a presumed graft-graft interaction, the HSC835 product predominated in 5 of 9 patients and resulted in sustained hematopoiesis for a median follow up of 303 days (range 140-401). The median time to neutrophil recovery (days to absolute neutrophil count of ≥500/uL) was shorter in recipients of HSC835 (i.e., 16 days [range, 6-23] versus 24 days [range, 22-30]) with the speed of neutrophil recovery correlating with the number of CD34+ cells infused (r2 = -0.87, p<0.05, Figure 1). With a median follow-up of 161 days (range, 40-401), six patients are alive. Primary causes of death were CMV pneumonitis, alveolar hemorrhage and interstitial pneumonitis in three patients. Conclusion The AHR antagonist SR1 is a potent inhibitor of HSC differentiation, resulting in marked expansion of HSPC. Hematopoietic reconstitution as early as day 6 is dependent on CD34+ cell dose in the expanded product, HSC835. Based on the long-term engraftment potential of HSC835 in half of the patients using the double UCB platform and the accelerated neutrophil recovery seen in patients recovering with HSC835, future patients will receive HSC835 alone eliminating the confounding graft-graft effects and potentially further reducing the time to neutrophil recovery. Disclosures: Wagner: Novartis: Research Funding. Brunstein:Novartis: Research Funding. McKenna:Novartis: Research Funding. Sumstad:Novartis: Research Funding. Maahs:Novartis: Employment. Boitano:Novartis: Employment. Cooke:Novartis: Employment. Bleul:Novartis: Employment.

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