Extracellular Tyrosyl-tRNA Synthetase Is a Potent Stimulator of Thrombocytopoiesis

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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Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.

Blood ◽

10.1182/blood.v120.21.2321.2321 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2321-2321

Author(s):

Brandon K Hadland ◽

Barbara Varnum-Finney ◽

Irwin D. Bernstein

Keyword(s):

Notch Signaling ◽

Ex Vivo ◽

Fetal Liver ◽

Signal Pathways ◽

Mouse Bone Marrow ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Notch Activation

Abstract Abstract 2321 An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC. Disclosures: Bernstein: Seattle Genetics, Inc.: Consultancy.

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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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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc Ca/Bcl-XL Plasma Cell Malignancy.

Blood ◽

10.1182/blood.v114.22.3835.3835 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3835-3835 ◽

Cited By ~ 1

Author(s):

Michael Fitzgerald ◽

Yueying Cao ◽

Bret Bannerman ◽

Zhi Li ◽

Olga Tayber ◽

...

Keyword(s):

Antitumor Activity ◽

Mouse Model ◽

Lymph Nodes ◽

Plasma Cell ◽

Research Funding ◽

Ex Vivo ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 3835 Poster Board III-771 Introduction The first generation proteasome inhibitor VELCADE® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMycCa/Bcl-XL mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMycCa/Bcl-XL mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMycCa/Bcl-XL PCM. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMycCa/Bcl-XL mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-XL (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMycCa/Bcl-XL tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM. Methods Cell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMycCa/Bcl-XL PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results In vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD50 values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The DP54-Luc disseminated mouse model of double transgenic iMycCa/Bcl-XL PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures: Cao: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

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Recombinant TAT-HOXB4 Protein Promotes Ex Vivo Expansion of Primitive Human Hematopoietic Cells.

Blood ◽

10.1182/blood.v104.11.2855.2855 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2855-2855

Author(s):

Gorazd Krosl ◽

Marie-Pier Giard ◽

Jana Krosl ◽

R. Keith Humphries ◽

Guy Sauvageau ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Cell Populations ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.

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Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion

Journal of Experimental Medicine ◽

10.1084/jem.20151563 ◽

2016 ◽

Vol 213 (9) ◽

pp. 1881-1900 ◽

Cited By ~ 83

Author(s):

Martin Chopra ◽

Marlene Biehl ◽

Tim Steinfatt ◽

Andreas Brandl ◽

Juliane Kums ◽

...

Keyword(s):

T Cells ◽

Cell Biology ◽

Acute Gvhd ◽

Ex Vivo ◽

Tumor Necrosis Factor Receptor ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Study Protocols

Donor CD4+Foxp3+ regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell–dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽

10.3390/cells10040730 ◽

2021 ◽

Vol 10 (4) ◽

pp. 730

Author(s):

Biji Mathew ◽

Leianne A. Torres ◽

Lorea Gamboa Gamboa Acha ◽

Sophie Tran ◽

Alice Liu ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Extracellular Vesicles ◽

Time Course ◽

Ganglion Cells ◽

Ex Vivo ◽

Dependent Manner ◽

Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

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Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

Frontiers in Immunology ◽

10.3389/fimmu.2021.668307 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paul D. Bates ◽

Alexander L. Rakhmilevich ◽

Monica M. Cho ◽

Myriam N. Bouchlaka ◽

Seema L. Rao ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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