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CD34 is a transmembrane phosphoglycoprotein used to selectively enrich bone marrow in hematopoietic stem cells for transplantation. Treating rats with CD34+ cells derived from human umbilical cord blood before or after heat stroke has been shown to promote survival. We investigated whether CD34- human placenta-derived stem cells (PDMSCs) could improve survival following heat stroke in rats. Rats were subjected to heat stress (42°C for 98 min) to induce heat stroke. Intravenous administration of PDMSCs 1 day before or immediately after the onset of heat stroke improved survival by 60{\%} and 20{\%}, respectively. Pre-treatment with CD34- PDMSCs protected against heat stroke injury more effectively than that treatment after injury. PDMSCs treatment attenuated cerebrovascular dysfunction, the inflammatory response, and lipid peroxidation. These data suggest human PDMSCs protect against heat stroke injury in rats. Moreover, these effects do not require the presence of CD34+ cells.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening, disease modeling, and heart repair. However, the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation, we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one, whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture, optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload, whereas twitch velocities decreased with afterload. Consequently, the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically, the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length, cardiomyocyte area and elongation, which are hallmarks of maturation. Furthermore, progressively increasing the level of afterload led to improved calcium handling, increased expression of several key markers of cardiac maturation, including a shift from fetal to adult ventricular myosin heavy chain isoforms. However, at the highest afterload condition, markers of pathological hypertrophy and fibrosis were also upregulated, although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together, our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs, while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload.

Targeted cancer immunotherapy with irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury, a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage, safety, and relative ease to isolate and culture. However, there has been a wide range in efficacy reported using MSC clinically and in preclinical models, likely due to differences in cell preparations and a significant amount of donor variability. In this study, we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC, one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI, an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed, the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment, suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430.

We hypothesized that the neonatal rat heart would bring transplanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to maturity as it grows to adult size. In neonatal rat heart, engrafted hiPSC derivatives developed partially matured myofibrils after 3 months, with increasing cell size and sarcomere length. There was no difference between grafts from hiPSC-CMs or hiPSC-derived cardiac progenitors (hiPSC-CPs) at 3 months, nor was maturation influenced by infarction. Interestingly, the infarcted adult heart induced greater human cardiomyocyte hypertrophy and induction of cardiac troponin I expression than the neonatal heart. Although human cardiomyocytes at all time points were significantly smaller than the host rat cardiomyocytes, transplanted neonatal rat cardiomyocytes reached adult size and structure by 3 months. Thus, the adult rat heart induces faster maturation than the neonatal heart, and human cardiomyocytes mature more slowly than rat cardiomyocytes. The slower maturation of human cardiomyocytes could be related to environmental mismatch or cell-autonomous factors.

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease of unknown etiopathogenesis with limited therapeutic options. IPF is characterized by an abundance of fibroblasts and loss of epithelial progenitors, which cumulates in unrelenting fibrotic lung remodeling and loss of normal oxygenation. IPF has been challenging to model in rodents; nonetheless, mouse models of lung fibrosis provide clues as to the natural progression of lung injury and remodeling, but many have not been useful in predicting efficacy of therapeutics in clinical IPF. We provide a detailed methodologic description of various iterations of humanized mouse models, initiated by the i.v. injection of cells from IPF lung biopsy or explants specimens into severe combined immunodeficiency (SCID)/beige or nonobese diabetic SCID gamma$ mice. Unlike cells from normal lung samples, IPF cells promote persistent, nonresolving lung remodeling in SCID mice. Finally, we provide examples and discuss potential advantages and pitfalls of human-specific targeting approaches in a humanized SCID model of pulmonary fibrosis.

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.

Transitioning CD19-directed chimeric antigen receptor (CAR) T cells from early-phase trials in relapsed patients to a viable therapeutic approach with predictable efficacy and low toxicity for broad application among patients with high unmet need is currently complicated by product heterogeneity resulting from transduction of undefined T-cell mixtures, variability of transgene expression, and terminal differentiation of cells at the end of culture. A phase 1 trial of 45 children and young adults with relapsed or refractory B-lineage acute lymphoblastic leukemia was conducted using a CD19 CAR product of defined CD4/CD8 composition, uniform CAR expression, and limited effector differentiation. Products meeting all defined specifications occurred in 93{\%} of enrolled patients. The maximum tolerated dose was 106 CAR T cells per kg, and there were no deaths or instances of cerebral edema attributable to product toxicity. The overall intent-to-treat minimal residual disease-negative (MRD-) remission rate for this phase 1 study was 89{\%}. The MRD- remission rate was 93{\%} in patients who received a CAR T-cell product and 100{\%} in the subset of patients who received fludarabine and cyclophosphamide lymphodepletion. Twenty-three percent of patients developed reversible severe cytokine release syndrome and/or reversible severe neurotoxicity. These data demonstrate that manufacturing a defined-composition CD19 CAR T cell identifies an optimal cell dose with highly potent antitumor activity and a tolerable adverse effect profile in a cohort of patients with an otherwise poor prognosis. This trial was registered at www.clinicaltrials.gov as {\#}NCT02028455.

Recent advances in the differentiation and production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) have stimulated development of strategies to use these cells in human cardiac regenerative therapies. A prerequisite for clinical trials and translational implementation of hPSC-derived CMs is the ability to manufacture safe and potent cells on the scale needed to replace cells lost during heart disease. Current differentiation protocols generate fetal-like CMs that exhibit proarrhythmogenic potential. Sufficient maturation of these hPSC-derived CMs has yet to be achieved to allow these cells to be used as a regenerative medicine therapy. Insights into the native cardiac environment during heart development may enable engineering of strategies that guide hPSC-derived CMs to mature. Specifically, considerations must be made in regard to developing methods to incorporate the native intercellular interactions and biomechanical cues into hPSC-derived CM production that are conducive to scale-up.