The reparative immunologic consequences of stem cell transplantation as a cellular therapy for refractory Crohn's disease.

Background: Treatment strategies for Crohn's disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous hematopoietic stem cell transplantation (SCT) has emerged as a therapy for medically refractory CD. SCT was developed to rescue cancer patients from myelosuppressive chemotherapy but its use for CD and other immune diseases necessitates reimagining SCT as a cellular therapy that restores appropriately responsive immune cell populations from hematopoietic progenitors in the stem cell autograft (i.e. immune "reset"). Here we present a paradigm to understand SCT as a cellular therapy for immune diseases and reveal how SCT re-establishes cellular immunity utilizing high-dimensional cellular phenotyping and functional studies of the stem cell grafts. Methods: Immunophenotyping using CyTOF, single cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing was performed on peripheral blood and intestinal tissue samples from refractory CD patients who underwent SCT. The stem cell graft from these patients was analyzed using flow cytometry and functionally interrogated using a murine model for engraftment. Results: Our study revealed a remodeling of intestinal macrophages capable of supporting mucosal healing that was independently validated using multimodal studies of immune reconstitution events including CyTOF and scRNA-seq. Functional interrogation of hematopoietic stem cells (HSCs) using a xenograft model demonstrated that HSCs shape the timing of immune reconstitution, the selected reconstitution of specific cell lineages and potentially the clinical efficacy of SCT. Conclusions: These studies indicate that SCT serves as a myeloid-directed cellular therapy re-establishing homeostatic intestinal macrophages that support intestinal healing and suggest refractory CD evolves from impairment of restorative functions in myeloid cells. Furthermore, we report heterogeneity among HSCs from CD patients which may drive SCT outcomes and suggests an unrecognized impact of CD pathophysiology on HSC in the marrow niche.

Multimodal single-cell analyses reveal mechanisms of perianal fistula in diverse patients with Crohn's disease.

BACKGROUND: Crohn's disease complicated by perianal fistulae is more prevalent and severe in patients of African ancestry. METHODS: We profiled single cells from diverse patients with Crohn's disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing. FINDINGS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn's genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints. CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula. FUNDING: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.

NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures.

OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Increased angiogenesis and improved left ventricular function after transplantation of myoblasts lacking the MyoD gene into infarcted myocardium.

Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD(-/-)), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD(-/-) myoblasts after transplantation into infarcted heart. We demonstrate that MyoD(-/-) myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD(-/-) and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD(-/-) myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD(-/-) myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD(-/-) myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation.

MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce induced pluripotent stem cells.

Expression of the four transcription factors, that is, Oct4, Sox2, cMyc, and Klf4 has been shown to generate induced pluripotent stem cells (iPSCs) from many types of specialized differentiated somatic cells. It remains unclear, however, whether fully committed skeletal muscle progenitor cells (myoblasts) have the potency to undergo reprogramming to develop iPSCs in line with previously reported cases. To test this, we have isolated genetically marked myoblasts derived from satellite cell of adult mouse muscles using the Cre-loxP system (Pax7-CreER:R26R and Myf5-Cre:R26R). On infection with retroviral vectors expressing the four factors, these myoblasts gave rise to myogenic lineage tracer lacZ-positive embryonic stem cell (ESC)-like colonies. These cells expressed ESC-specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of myoblast-derived iPSCs. Continuous expression of the MyoD gene, a master transcription factor for skeletal muscle specification, inhibited this reprogramming process in myoblasts. In contrast, reprogramming myoblasts isolated from mice lacking the MyoD gene led to an increase in reprogramming efficiency. Our data also indicated that Oct4 acts as a transcriptional suppressor of MyoD gene expression through its interaction with the upstream enhancer region. Taken together, these results indicate that suppression of MyoD gene expression by Oct4 is required for the initial reprogramming step in the development of iPSCs from myoblasts. This data suggests that the skeletal muscle system provides a well-defined differentiation model to further elaborate on the effects of iPSC reprogramming in somatic cells.

MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3.

The molecules that regulate the apoptosis cascade are also involved in differentiation and syncytial fusion in skeletal muscle. MyoD is a myogenic transcription factor that plays essential roles in muscle differentiation. We noticed that MyoD(-/-) myoblasts display remarkable resistance to apoptosis by down-regulation of miR-1 (microRNA-1) and miR-206 and by up-regulation of Pax3. This resulted in transcriptional activation of antiapoptotic factors Bcl-2 and Bcl-xL. Forced MyoD expression induces up-regulation of miR-1 and miR-206 and down-regulation of Pax3, Bcl-2, and Bcl-xL along with increased apoptosis in MyoD(-/-) myoblasts. In contrast, MyoD gene knockdown increases cell survival of wild-type myoblasts. The 3' untranslated region of Pax3 mRNA contains two conserved miR-1/miR-206-binding sites, which are required for targeting of these microRNAs (miRNAs). Therefore, these data suggest that MyoD not only regulates terminal differentiation but also apoptosis through miRNA-mediated down-regulation of Pax3. Finally, MyoD, miR-1, and miR-206 are all down-regulated in quiescent satellite cells, which may be required for maintenance of muscle stem cells.

Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor, and therefore both homozygous (Flt-1(-/-)) and heterozygous (Flt-1(+/-)) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1(+/-) and mdx:Flt-1(+/-) adult mice also display a developmentally increased vascular density in skeletal muscle compared with the wild-type and mdx mice, respectively. The mdx:Flt-1(+/-) mice show improved muscle histology compared with the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1(+/-) mice have an increase in muscle blood flow and force production, compared with the mdx mice. Consequently, the mdx:utrophin(-/-):Flt-1(+/-) mice display improved muscle histology and significantly higher survival rates compared with the mdx:utrophin(-/-) mice, which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.