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. SAVI patients are affected by systemic abnormal inflammation involving skin, blood vessels and lungs. This syndrome is caused by a de novo mutation in the TMEM173 gene, which leads to constitutive STING activation. We hypothesize that STING overactivation drives a chronic activation of the interferon pathway and contributes to endothelial cell dysfunction in SAVI. To understand this, we developed patient-specific iPSCs and their derivative endothelial cell (SAVI-iEC) lines as a model of SAVI. When comparing SAVI-iECs to healthy control iPSCs, cells derived from SAVI patients gradually lost EC characteristics and acquired the features of mesenchymal cells. This defect can be rescued in iECs derived from isogeneic control iPSCs generated by using Crispr-cas9 gene correction in the SAVI-iPSC. This result demonstrates that STING overactivation in SAVI spontaneously promotes the biological process of the endothelia to mesenchymal cell transition (Endo-MT). Additionally, gene expression profile and pathway analysis shows that the Janus Kinases-Signal Transducer and Activator of Transcription protein (JAK-STAT) pathway might be involved in this process and STAT3 may play an important role to mediate Endo-MT. 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 Khlmeier-Degos. as well as for other rare conditions with vascular phenotype that may have unclear genetic cause or pathomechanism.