Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5– donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell–mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.
Isaac M. Barber-Axthelm, Valerie Barber-Axthelm, Kai Yin Sze, Anjie Zhen, Gajendra W. Suryawanshi, Irvin S.Y. Chen, Jerome A. Zack, Scott G. Kitchen, Hans-Peter Kiem, Christopher W. Peterson
Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased co-enrichment of epidermal growth factor receptor (EGFR) and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that co-inheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with co-enriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.
Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha S. Anand, Bjarne Winther Kristensen, Justin Lathia
Resident vascular adventitial SCA1(+) progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling are significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM-specific expression of Gli1, we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell-specific Klf4 depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a pro-fibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis, but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable anti-fibrotic approach.
Sizhao Lu, Austin J. Jolly, Keith A. Strand, Allison M. Dubner, Marie F. Mutryn, Karen S. Moulton, Raphael A. Nemenoff, Mark W. Majesky, Mary C.M. Weiser-Evans
Chronic kidney disease (CKD) induces the failure of arteriovenous fistulas (AVF) and promotes the differentiation of vascular adventitial GLI1+ mesenchymal stem cells (GMCs). However, the roles of GMCs in forming neointima in AVFs remains unknown. GMCs isolated from CKD mice showed increased potential capacity of differentiation into myofibroblast-like cells. Increased activation of expression of PDGFRA and hedgehog (HH) signaling were detected in adventitial cells of AVFs from ESRD patients and CKD mice. PDGFRA was translocated and accumulated in early endosome when hedgehog signaling stimulates was activated. In endosome, PDGFRA mediated activation of TGFB1/SMAD signaling promoting GMCs differentiation into myofibroblast, extracellular matrix deposition, and vascular fibrosis. These responses resulted in neointima formation and AVF failure. Knockout (KO) of Pdgfra or inhibition of HH signaling in GMCs suppressed the differentiation of GMCs into myofibroblasts. In vivo, specific KO of Pdgfra inhibited GMC activation and vascular fibrosis, resulting in suppression of neointima formation and improvement of AVF patency despite CKD. Our findings could yield strategies for maintaining AVF functions.
Ke Song, Ying Qing, Qunying Guo, Eric K. Peden, Changyi Chen, William E. Mitch, Luan Truong, Jizhong Cheng
The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required Cdc42-mediated growth response and Cdc42-deficient enteroids undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiological, but non-conventionally, spliced Cdc42 variant 2 (V2), exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration while elevating this signaling cascade is capable of initiating protection from genotoxic injury.
Xiao Zhang, Sheila Bandyopadhyay, Leandro P. Araujo, Kevin Tong, Juan Flores, Daniel Laubitz, Yanlin Zhao, George Yap, Jingren Wang, Qingze Zou, Ronaldo P. Ferraris, Lanjing Zhang, Wenwei Hu, Edward M. Bonder, Pawel R. Kiela, Robert J. Coffey, Michael Verzi, Ivaylo I. Ivanov, Nan Gao
Ribosomopathies are congenital disorders caused by mutations in the genes encoding ribosomal and other functionally related proteins. They are characterized by anemia, other hematopoietic and developmental abnormalities, and p53 activation. Ribosome assembly requires coordinated expression of many ribosomal protein (RP) genes; however, the regulation of RP gene expression, especially in hematopoietic stem cells (HSCs), remains poorly understood. MYSM1 is a transcriptional regulator essential for HSC function and hematopoiesis. We established that HSC dysfunction in Mysm1 deficiency is driven by p53; however, the mechanisms of p53 activation remained unclear. Here, we describe the transcriptome of Mysm1-deficient mouse HSCs and identify MYSM1 genome-wide DNA binding sites. We establish a direct role for MYSM1 in RP gene expression and show a reduction in protein synthesis in Mysm1–/– HSCs. Loss of p53 in mice fully rescues Mysm1–/– anemia phenotype but not RP gene expression, indicating that RP gene dysregulation is a direct outcome of Mysm1 deficiency and an upstream mediator of Mysm1–/– phenotypes through p53 activation. We characterize a patient with a homozygous nonsense MYSM1 gene variant, and we demonstrate reduced protein synthesis and increased p53 levels in patient hematopoietic cells. Our work provides insights into the specialized mechanisms regulating RP gene expression in HSCs and establishes a common etiology of MYSM1 deficiency and ribosomopathy syndromes.
Jad I. Belle, HanChen Wang, Amanda Fiore, Jessica C. Petrov, Yun Hsiao Lin, Chu-Han Feng, Thi Tuyet Mai Nguyen, Jacky Tung, Philippe M. Campeau, Uta Behrends, Theresa Brunet, Gloria Sarah Leszinski, Philippe Gros, David Langlais, Anastasia Nijnik
In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage–sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially novel insights into hematopoiesis and reveal DMHCA’s mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.
Cristiano P. Vieira, Seth D. Fortmann, Masroor Hossain, Ana Leda Longhini, Sandra S. Hammer, Bright Asare-Bediako, David K. Crossman, Micheli S. Sielski, Yvonne Adu-Agyeiwaah, Mariana Dupont, Jason L. Floyd, Sergio Li Calzi, Todd Lydic, Robert S. Welner, Gary J. Blanchard, Julia V. Busik, Maria B. Grant
Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin resulting in pathologic cartilage and bone matrix deposition. CCN family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1(also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1/CCN4 expression across traumatic and genetic HO mouse models, as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1 null tenocytes revealed enrichment in signaling pathways such as STAT3 and PCP signaling that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase A. Pagani, Charles Hwang, David M. Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading, and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ), and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared to the wild type Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically-stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage, and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.
Jonathan P. Gumucio, Martin M. Schonk, Yalda A. Kharaz, Eithne Comerford, Christopher L. Mendias
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. When human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patients’ hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these two hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study with hiPSC-derived cardiomyocytes revealed that fenofibrate could inhibit mitochondria-induced apoptosis in the DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified two promising small molecules with in vivo efficacy.
Congshan Sun, In Young Choi, Yazmin I. Rovira Gonzalez, Peter Andersen, C. Conover Talbot Jr., Shama R. Iyer, Richard M. Lovering, Kathryn R. Wagner, Gabsang Lee
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