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Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-gamma$ (IFN-gamma$)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-gamma$ is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-gamma$ within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-gamma$ via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-gamma$-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-gamma$-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-gamma$-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-gamma$ within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Mesenchymal stem cells (MSCs) have been isolated from various mesodermal and ectodermal tissues. While the phenotypic and functional heterogeneity of MSCs stemming from their developmental origins has been acknowledged, the genetic and environmental factors underpinning these differences are not well-understood. Here, we investigated whether substrate stiffness mediated mechanical cues can directly modulate the development of ectodermal MSCs (eMSCs) from a precursor human neural crest stem cell (NCSC) population. We showed that NCSC-derived eMSCs were transcriptionally and functionally distinct from mesodermal bone marrow MSCs. eMSCs derived on lower substrate stiffness specifically increased their expression of the MSC marker, CD44 in a Rho-ROCK signaling dependent manner, which resulted in a concomitant increase in the eMSCs' adipogenic and chondrogenic differentiation potential. This mechanically-induced effect can only be maintained for short-term upon switching back to a stiff substrate but can be sustained for longer-term when the eMSCs were exclusively maintained on soft substrates. We also discovered that CD44 expression modulated eMSC self-renewal and multipotency via the downregulation of downstream platelet-derived growth factor receptor beta (PDGFRbeta$) signaling. This is the first instance demonstrating that substrate stiffness not only influences the differentiation trajectories of MSCs but also their derivation from upstream progenitors, such as NCSCs.

Immunomodulation contributes to the antitumor efficacy of the fractionated radiation therapy (RT). Here, we describe immune effects of RT with concurrent systemic cisplatin or cetuximab treatment of patients with stage III-IV head and neck squamous cell carcinoma (HNSCC). Using longitudinally collected blood samples, we identified significant changes in cytokines/chemokines and immune cell populations compared to immune-related gene expression profiles in peripheral blood mononuclear cells (PBMCs). The 7-week combinatorial RT resulted in gradual elevation of proinflammatory mediators (IFNgamma$, IL-6, TNFɑ, CCL2), while levels of IL-12, cytokine essential for antitumor immune responses, were decreased. These effects correlated with progressive accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) with detectable activity of STAT3 and PD-L1 expression, underscoring tolerogenic effects of MDSCs. Correspondingly, gene expression analysis of PBMCs harvested after two weeks of combinatorial RT, found upregulation of several immunosuppressive mediators. These included IL6, IL6R, STAT3 and PDL1, which could represent IL-6/STAT3-driven tolerogenic signaling, which inhibits T cell and NK activity. Overall, our results suggest that potential immunostimulatory effects of combinatorial RT in HNSCC patients are likely limited by tolerogenic STAT3 signaling and PD-L1 upregulation in myeloid immune cells. Further studies will clarify whether STAT3 targeting could augment RT efficacy and durability of antitumor responses.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM) who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7). Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry the pathogenic MYH7 variant p.Val698Ala, have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) obtained from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM). PBMCs were reprogrammed to a pluripotent state following transfection with non-integrative episomal vectors carrying reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotency markers, possess trilineage differentiation potential, carry rare variants identified in DNA isolated directly from the patient's whole blood, have a normal karyotype and no longer carry episomal vectors for reprogramming. This line is a useful resource for identifying unknown genetic causes of HCM.

The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with on-target off-tumor" activity. Here

Pluripotent stem cell-derived cardiomyocyte grafts can remuscularize substantial amounts of infarcted myocardium and beat in synchrony with the heart, but in some settings cause ventricular arrhythmias. It is unknown whether human cardiomyocytes can restore cardiac function in a physiologically relevant large animal model. Here we show that transplantation of ∼750 million cryopreserved human embryonic stem cell-derived cardiomyocytes (hESC-CMs) enhances cardiac function in macaque monkeys with large myocardial infarctions. One month after hESC-CM transplantation, global left ventricular ejection fraction improved 10.6 ± 0.9{\%} vs. 2.5 ± 0.8{\%} in controls, and by 3 months there was an additional 12.4{\%} improvement in treated vs. a 3.5{\%} decline in controls. Grafts averaged 11.6{\%} of infarct size, formed electromechanical junctions with the host heart, and by 3 months contained ∼99{\%} ventricular myocytes. A subset of animals experienced graft-associated ventricular arrhythmias, shown by electrical mapping to originate from a point-source acting as an ectopic pacemaker. Our data demonstrate that remuscularization of the infarcted macaque heart with human myocardium provides durable improvement in left ventricular function.

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with {\textgreater}85{\%} purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.