iPS-technology and patient specific disease models

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

The development and application of iPSC technology promises to revolutionize our understanding of disease mechanisms and improve the availability of treatment options. The ability to generate patient specific iPSC allows for the development of cell-based disease modeling where it has previously been extremely difficult to obtain sufficient amounts of the relevant human cell types to study such as cardiomyocytes, mesenchymal stem cells (MSCs), endothelial (ECs) and vascular smooth muscle cells (VSMCs). Further, this technology provides a powerful platform for investigating the mechanisms in rare genetic disorders underlying cardiovascular diseases. We have to-date successfully generated a human iPSC biobank with several samples derived from healthy volunteers and patients carrying genetic defects for conditions such as Arterial Calcifications due to Deficiency in CD73 (ACDC), Autosomal Dominante Hyper-immunoglobulin E Syndromes (AD-HIES/Jobs syndrome), Stimulator of Interferon Genes (STING)-Associated Vasculopathy with onset in Infancy (SAVI), Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), Deficiency of Adenosine Deaminase 2 (DADA2), OTULIN-Related Autoinflammatory Syndrome (ORAS), Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature (CANDLE), Turner Syndrome (TS) and Khlmeier-Degos (K-D). The majority of iPSC lines have also been extensively characterized to exhibit the potential of unlimited self-renewal as well as the ability to differentiate into three germ layers. Additionally, we have established novel differentiation protocols with efficient, stage-wise, chemically-defined strategies for short-term induction of mesoderm lineage cells (ECs, MSCs, VSMCs, Hematopoietic lineage cells). In addition, in collaboration with Dr. Wimmer (Institute of Molecular Biotechnology of the Austrian Academy of Sciences), we have developed an approach for generating human blood vessel organoids. This approach has proven critical in increasing our understanding of the disease pathophysiology as well as allowed for identifying drugs/small molecules as potential novel therapeutic strategies or cell replacement therapy. Using these technologies with ACDC, we have demonstrated a compensatory upregulation of TNAP leading to an insufficient production of adenosine and a marked decrease in PPi, causing the vascular calcifications in ACDC samples from patients in vitro. In vivo, we also identified an inhibitory pathway downstream of the A2B adenosine receptor signaling pathway and several therapeutic drug targets, with etidronate being the most clinically viable candidate. On the other hand, when focusing on the underlying mechanisms of AD-HIES that is caused by loss-of-function mutations in signal transducer and activator of transcription 3 (STAT3), we were able to identify that STAT3 plays a critical role in reprogramming of somatic cells towards iPSCs through activation of Nanog. We also developed a teratoma model (murine) system with iPSC to explore pharmacological targets for treatment of AD-HIES. Deficiency of ADA2 (DADA2) is a rare genetic disorder caused by mutations in the adenosine deaminase 2 gene, leading to vasculitis, systemic inflammation, early-onset stroke, immunodeficiency, and bone marrow failure. Despite a decade of research, the mechanism remains unclear due to the absence of suitable model systems, as ADA2 is not expressed in mice. We generated iPSCs from DADA2 patients to create blood vessel organoids and monocytes. These monocytes preferentially polarize to proinflammatory M1 macrophages and damage endothelial cells in co-culture studies. SAVI syndrome, caused by a de novo TMEM173 mutation, results in chronic inflammation affecting the skin, blood vessels, and lungs due to STING overactivation. We developed patient-specific iPSCs and endothelial cell lines (SAVI-iECs) to study this condition. SAVI-iECs showed a loss of endothelial characteristics and transition to mesenchymal cells, a change that can be corrected by gene editing. This transition appears driven by STING overactivation and involves the JAK-STAT pathway, with STAT3 playing a key role. While SAVI patients often experience pulmonary fibrosis, transgenic mice with STING mutations only exhibit lung inflammation. To address this, we created vascularized pulmonary organoids from SAVI patient iPSCs to better understand the fibrosis mechanism. In addition to conventional monolayer cultures, we have established a suite of advanced disease modeling platforms, including vascular organoids, immune-vascular organoids, vascularized lung organoids, and vascularized brain organoids, to capture the complex manifestations of rare vascular diseases. Among these, immune-vascular organoids are particularly powerful, as they incorporate both vascular cell types and diverse myeloid populations, thereby enabling the study of vascular–immune interactions in a physiologically relevant context. Using this system, we showed that the organoids faithfully recapitulate disease phenotypes observed in patients with STING-associated vasculopathy with onset in infancy (SAVI) and Lyn kinase-associated vasculopathy and liver fibrosis (LAVLI). Single-cell RNA sequencing (scRNA-seq) further revealed shared molecular alterations across these conditions, most notably the dysregulation of the integrated stress response (ISR). This finding suggests that ISR activation may represent a convergent pathogenic mechanism in distinct autoinflammatory vasculopathies. Strikingly, pharmacological inhibition of ISR signaling led to a marked reduction in inflammatory cytokine production in both SAVI and LAVLI organoids, highlighting ISR as a potential therapeutic target for these otherwise intractable diseases. K-D is a rare small vessel vasculopathy of unknown etiology leading to small blood vessel occlusions in multiple organs, including the skin, central nervous system, eye, gastrointestinal tract, lungs, and heart. K-D commonly presents as a benign cutaneous form with lesions that appear as erythematous papules and evolve to form a scar with an atrophic, porcelain-white center surrounded by a telangiectatic border. Progression to systemic K-D, or malignant atrophic papulosis, occurs in 2/3 of the patients, is often debilitating, and can be fatal (60-70% mortality rate within 2-5 years after diagnosis) due to GI perforations, brain infarcts, spinal lesions, cardiac or pulmonary failure, sepsis or cachexia. We plan to investigate the mechanism of vasculopathy in K-D patients using iPSC-derived patient-specific in vitro and in vivo disease models that are well-established platforms in our team. We have submitted PBMCs from 5 patients with systemic K-D disease to the NHLBI iPSC Core facility, and we plan to induce endothelial cells (iECs) and vascular and smooth muscle cells (iVSMCs) to use in experiments to investigate the interaction of patient-specific iECs /iVSMC with patients' PBMCs and isolated CD8 T-cells in an in vitro co-culture assay and determine cell type-specific transcriptional profile on a single cell level. The research accomplished with these iPSC lines and their derivatives has led to a large volume of publications in Stem Cell Research, and Chemical Research in Toxicology over the last year. We plan to continue to apply these strategies to study the genetic cause and disease mechanisms with on-going research for SAVI, DADA2, CADASIL, CANDLE and Köhlmeier-Degos. as well as for other rare conditions with vascular phenotype that may have unclear genetic cause or pathomechanism.