Development of in vitro and in vivo vascular disease models

We have successfully established a powerful patient-specific disease-modeling platform to explore disease mechanisms and novel therapy targets using human induced pluripotent stem cells (hiPSCs) and their derivatives. We have further established novel differentiation protocols for efficient, stage­wise, chemically defined strategy for the induction of mesoderm lineage cells, including endothelial cells (iECs), mesenchymal stem cells (iMSCs), vascular smooth muscle cells (iVSMCs), hematopoietic stem cells (iHSCs), and myeloid lineage cells (iMLCs). 

This versatile system can be used for screening approaches, including high throughput sequencing, loss- and gain-of-function studies, and in vitro/in vivo disease modeling to identify disease-causing genetic variants and associated signaling pathways. Patient-specific hiPSCs and their derivatives can be further used to test drugs or small molecules for the development of new therapeutic strategies in culture or in-vivo models such well-established teratoma and/or 3D organoid systems.

Through a variety of murine models, including vein grafting, arterial wire injury, hindlimb ischemia and myocardial infarction, we can investigate vascular remodeling mechanisms as well as the effects of biochemical/iPSC therapies. This approach is complemented with the use of a wide range of cell culture lines (standard cell lines, patient-derived primary and iPSCs) and cutting-edge techniques of co-culture and 3D printed structures in parallel with large scale Omics studies including single cell RNA sequencing, proteomics and metabolomics.

in vitro in vivo models
Schematic showing how patient samples are processed to develop patient-specific iPSC and derived cell lines such as induced endothelial (iEC), smooth muscle (iSMC), mesenchymal (iMSC), monocyte (iMono) and dendritic (iDC) cells. These can be further used in teratoma and vascular organoid (VO) models of disease and they can also be genetically corrected to develop isogenic controls.

Researchers: Guibin, Quan


  1. Zhen Yu, Natalia I Dmitrieva, Avram D. Walts, Hui Jin, Yangtengyu Liu, Xianfeng Ping, Elisa A. Ferrante, Lugui Qiu, Steven M. Holland, Alexandra F. Freeman, Guibin Chen, Manfred Boehm. STAT3 modulates reprogramming efficiency of human somatic cells; Insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations. Biology Open, 2020 bio.052662 Doi:10.1242,bio.052662.
  2. Pei-Hsuan Chu, Guibin Chen, David Kuo, John Braisted, Ruili Huang, Yuhong Wang, Anton Simeonov, Manfred Boehm, and David L. Gerhold A Stem Cell-Derived Endothelial Cell Model that Responds to Tobacco Smoke Like Primary Endothelial Cells. Chemical Research in Toxicology, 2020 (33):751-763.
  3. Guibin Chen, Zhongwen Li, Yangtengyu Liu, Daniel Chen, Jeanette Beers, Cornelia Cudrici, Elisa A. Ferrante, Robin Schwartzbeck, Natalia Dmitrieva, Dan Yang, Jizhong Zou, M. Luisa Iruela-Arispe, and Manfred Boehm. Generation of human induced pluripotent stem cells (NIHTVBi004-A, NIHTVBi005-A, NIHTVBi006-A, NIHTVBi007-A, NIHTVBi008-A) from CADASIL patients withNOTCH3 mutation. Stem Cell Research, 2020 (45):101821.
  4. Juan Pablo Ruiz, Guibin Chen, Juan Jesus Haro Mora, Keyvan Keyvanfar,Chengyu Liu, Jizhong Zou, Jeanette Beers, Hanan Bloomer, Husam Qanash, Naoya Uchida, John F.Tisdale, Manfred Boehm, Andre Larochelle. Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system. Stem Cell Research, 2019 (41), 101600.
  5. Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, Lindenhofer D, Chen G, Boehm M, Agu CA, Yang F, Fu B, Zuber J, Knoblich JA, Kerjaschki D, Penninger JM. Human blood vessel organoids as a model of diabetic vasculopathy. Nature. 2019; doi: 10.1038/s41586-018-0858-8

Arterial Calcification due to Deficiency of CD73 (ACDC)

The Cardiovascular Regenerative Medicine Lab was part of the team who identified the genetic cause of ACDC in 2011. This is an autosomal recessive disorder caused by biallelic mutations in the gene ecto-5-prime-nucleotidase (NT5E) encoding CD73, a protein that functions in extracellular ATP metabolism converting AMP to adenosine. ACDC patients develop arterial calcifications in the lower limbs as well as small joint capsules of the hands and feet. Primary fibroblasts from ACDC individuals show markedly reduced expression of NT5E mRNA, CD73 protein and complete loss of enzyme activity.

ACDC patient-specific iPSC and their derived iMSCs display increased alkaline phosphatase activity and can become calcified both in vivo and in vitro. They also display a compensatory upregulation of TNAP leading to insufficient production of adenosine and a marked decrease in pyrophosphate (PPi), which likely causes the vascular calcifications observed in ACDC patients. Using an ACDC patient-specific hiPSC model, we were able to identify an inhibitory pathway downstream of the A2B adenosine receptor signaling pathway and several therapeutic drug targets that led to a treatment clinical study in ACDC patients with sodium etidronate.

Schematic showing how patient samples are processed to develop patient-specific iPSC and derived cell lines such as induced endothelial (iEC), smooth muscle (iSMC), mesenchymal (iMSC), monocyte (iMono) and dendritic (iDC) cells. These can be further used in teratoma and vascular organoid (VO) models of disease and they can also be genetically corrected to develop isogenic controls.


  1. St Hilaire C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F, Carlson-Donohoe H, Lederman RJ, Chen MY, Yang D, Siegenthaler MP, Arduino C, Mancini C, Freudenthal B, Stanescu HC, Zdebik AA, Chaganti RK, Nussbaum RL, Kleta R, Gahl WA, Boehm M..NT5E mutations and arterial calcifications. N Engl J Med, 2011. 364(5): p. 432-42.
  2. Jin H, St Hilaire C, Huang Y, Yang D, Dmitrieva NI, Negro A, Schwartzbeck R, Liu Y, Yu Z, Walts A, Davaine JM, Lee DY, Donahue D, Hsu KS, Chen J, Cheng T, Gahl W, Chen G, Boehm M. Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC. Sci Signal, 2016. 9(458): p. ra121.
  3. Lakshmipathy DR, Cudrici CD, Dyda F, Xu W, Ferrante EA, Nguyen DT, Carney KM, Rollison S, Chen MY, Nesti LJ, Boehm M, Brofferio A, Wen H.., Morphology and Chemical Identity of Periarticular and Vascular Calcification in a Patient with the Rare Genetic Disease of Arterial Calcification Due to Deficiency of CD73 (ACDC). Radiology Case Reports, 2020. 15(10): p. 1883-1886.
  4. Cudrici C, Ferrante E , Manfred Boehm M: Basic molecular mechanism of vascular calcification. Coronary Calcium: A Comprehensive Understanding of Its Biology, Use in Screening, and Interventional Management 1st Editions, Edited by Aloke Virmani Finn, 2019

Autosomal dominant hyper-IgE syndrome (AD-HIES; Job’s syndrome)

AD-HIES is a rare immunodeficiency affecting fewer than 1 per million people worldwide that presents early in childhood with multiple life-threatening infections, such as recurrent staphylococcal abscesses and pyogenic pneumonias. Later in life, other abnormalities outside the immune system become evident. As opposed to complete tissue healing experienced by otherwise healthy patients after pneumonia, aberrant healing often occurs in AD-HIES patients, gradually destroying the lung parenchyma. Additionally, skeletal and connective tissue abnormalities including facial dysmorphism, osteoporosis, bone fractures, scoliosis and joint hyperextensibility are frequent in AD-HIES patients. Vascular abnormalities include arterial tortuosity and abnormal dilatation and aneurysms of medium sized arteries, all of which can lead to potentially fatal complications such as myocardial infarction and subarachnoid hemorrhage. 

The mystery surrounding the multisystem presentation of AD-HIES was partially clarified when, in 2007, the genetic cause of AD-HIES was identified as dominant-negative mutations in the STAT3 gene. This transcriptional factor has multiple targets playing crucial roles in basic cellular functions, and is widely expressed in multiple tissues and representing a major hub integrating and transducing signaling withing and between intracellular signaling pathway [1].

By applying patient specific disease modeling, next generation sequencing and signaling pathway analysis, we revealed major abnormalities in angiogenic, tissue remodeling and wound repair responses in AD-HIES patients driven by deficiencies in HIF1α signaling [2, 3, 4]. By demonstrating therapeutic benefit of HIF1α stabilizing drugs in these models, we identified a novel treatment option that is currently in late development stages for renal anemia, which can also be used for this rare disease off-label [4].

AD-HIES; Job's Syndrome


  1. Steven M. Holland, M.D., Frank R. DeLeo, Ph.D., Houda Z. Elloumi, Ph.D., Amy P. Hsu, B.A., Gulbu Uzel, M.D., Nina Brodsky, B.S., Alexandra F. Freeman, M.D., Andrew Demidowich, B.A., Joie Davis, A.P.R.N., Maria L. Turner, M.D., Victoria L. Anderson, C.R.N.P., Dirk N. Darnell, M.A., Pamela A. Welch, B.S.N., Douglas B. Kuhns, Ph.D., David M. Frucht, M.D., Harry L. Malech, M.D., John I. Gallin, M.D., Scott D. Kobayashi, Ph.D., Adeline R. Whitney, B.A., Jovanka M. Voyich, Ph.D., James M. Musser, M.D., Ph.D., Cristina Woellner, M.Sc., Alejandro A. Schäffer, Ph.D., Jennifer M. Puck, M.D., and Bodo Grimbacher, M.D. STAT3 Mutations in the Hyper-IgE Syndrome.; N Engl J Med, 2007; 357:1608-1619. DOI: 10.1056/NEJMoa073687
  2. Hui Jin, Zhen Yu, Keron Navarengom, Yangtengyu Liu, Natalia Dmitrieva, Amy P. Hsu, Robin Schwartzbeck, Cornelia Cudrici, Elisa A. Ferrante, Dan Yang, Steven M. Holland, Alexandra F. Freeman, Manfred Boehm, and Guibin Chen. Generation of human induced pluripotent stem cell lines (NIHTVBi011-A, NIHTVBi012-A, NIHTVBi013-A) from autosomal dominant Hyper IgE syndrome (AD-HIES) patients carrying STAT3 mutation. Stem Cell Research, 2019 (41), 101586. DOI: 10.1016/j.scr.2019.101586
  3. Yu Z, Dmitrieva NI, Walts AD, Jin H, Liu Y, Ping X, Ferrante EA, Qiu L, Holland SM, Freeman AF, Chen G, Boehm M (2020) STAT3 modulates reprogramming efficiency of human somatic cells; Insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations.; Biology Open (2020) 9, bio052662.
  4. Natalia I. Dmitrieva, Avram D. Walts, Dai P. Nguyen, Alex Grubb, Xue Zhang, Xujing Wang, Xianfeng Ping, Hui Jin, Zhen Yu, Zu-Xi Yu, Dan Yang, Robin Schwartzbeck, Clifton L. Dalgard, Beth A. Kozel, Mark D. Levin, Russell H. Knutsen, Delong Liu, Joshua D. Milner, Diego B. López, Michael P. O'Connell, Chyi-Chia R. Lee, Ian A. Myles, Amy P. Hsu, Alexandra F. Freeman, Steven M. Holland, Guibin Chen, and Manfred Boehm. Impaired angiogenesis and extracellular matrix metabolism in Autosomal-Dominant Hyper-IgE Syndrome.; J Clin Invest. 2020;130(8):4167-4181.;

STING-associated vasculopathy with onset in infancy (SAVI)

SAVI is a gain-of-function of TMEM173-encoded protein STING associated autoimmune and vascular disease, in which patients present with early-onset (as early as 8 weeks of age) systemic inflammation, cutaneous vasculopathy, and pulmonary fibrosis. STING is a single string DNA sensor and is expressed not only by immune cells but also by vascular endothelial cells. Our studies have shown that overactivation of STING leads to chronic activation of the interferon pathway and endothelial dysfunction. Further, we observed micro thrombotic vascular changes in chronic skin lesions from patients, with vascular inflammation present in capillaries but not medium or large size vessels. We also found that markers of endothelial inflammation (inducible nitric oxide synthase), coagulation (tissue factor), and endothelial-cell adhesion and activation (E-selectin and intercellular adhesion molecule 1) were upregulated in vascular endothelial cells in SAVI patients. This points to a STING-induced EC dysfunction mechanism that may instigate an inflammatory and vaso-occlusive process localized to specific vascular beds.

The mechanisms of how STING activation regulates endothelial cell (EC) function and how EC dysfunction contributes to patient phenotype, particularly interstitial lung fibrosis, remain currently unknown and are our main focus. We have generated SAVI patient-specific iECs for an in vitro disease model, in which we have established endothelial dysfunction phenotype and have successfully rescued endothelial function by CRISPR CAS9 mediated gene editing strategy. We are currently working on high throughput sequencing data analysis;to furth reveal the signaling pathways leading to pulmonary fibrosis and we hope to identify potential therapies for clinical trials for SAVI patients.



  1. Liu Y*, Jesus AA*, Marrero B*, Yang D, Ramsey SE, Montealegre Sanchez GA, Tenbrock K, Wittkowski H, Jones OY, Kuehn HS, Lee CC, DiMattia MA, Cowen EW, Gonzalez B, Palmer I, DiGiovanna JJ, Biancotto A, Kim H, Tsai WL, Trier AM, Huang Y, Stone DL, Hill S, Kim HJ, St Hilaire C, Gurprasad S, Plass N, Chapelle D, Horkayne-Szakaly I, Foell D, Barysenka A, Candotti F, Holland SM, Hughes JD, Mehmet H, Issekutz AC, Raffeld M, McElwee J, Fontana JR, Minniti CP, Moir S, Kastner DL, Gadina M, Steven AC, Wingfield PT, Brooks SR, Rosenzweig SD, Fleisher TA, Deng Z, Boehm M, Paller AS, Goldbach-Mansky R. Activated STING in a vascular and pulmonary syndrome. N Engl J Med. 2014 Aug 7; 371(6):507-18.
  2. Mona Motwani , Scott Pesiridis , Katherine A Fitzgerald. DNA sensing by the cGAS-STING pathway in health and disease. Nat Rev Genet 2019 Nov;20(11):657-674 doi: 10.1038/s41576-019-0151-1.
  3. Hannah Yang , Won Suk Lee , So Jung Kong , Chang Gon Kim , Joo Hoon Kim , Sei Kyung Chang , Sewha Kim, Gwangil Kim, Hong Jae Chon , Chan Kim. STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade J Clin Invest 2019 Jul 25;129(10):4350-4364. doi: 10.1172/JCI125413.

Deficiency of Adenosine Deaminase 2 (DADA2)

DADA2 is an autoinflammatory disease caused by a loss-of-function mutation of ADA2 encoded by the CECR1 (Cat Eye Syndrome Chromosome Region, Candidate 1) gene. DADA2 patients present with intermittent fevers, recurrent lacunar strokes, livedoid rash, and bone marrow failure starting in early childhood. ADA2 is a low-affinity enzyme that degrades extracellular adenosine to inosine and plays an unknown growth factor role to regulate T cell function. ADA2 is primarily expressed and secreted by myeloid cells but its role in human autoinflammatory vasculopathy is unknown. 

We have previously found that TNFa is significantly increased in DADA2 patients and anti-TNF treatment may help reduce strokes in DADA2 patients. How the loss of ADA2 function in myeloid cells leads to vascular endothelial injury and to strokes remains far from clear. We have generated DADA2 patient-specific iMLCs and have differentiated them to different macrophage subtypes (M0, M1 and M2) by the M-CSF method in order to develop an in vitro disease model to further investigate the disease mechanisms. We continue to use this iPSC platform to investigate how myeloid/endothelial cels interact to mediate vascular injury in DADA2 patients.



  1. Zhou Q*, Yang D*, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, Stone DL, Chae JJ, Rosenzweig SD, Bishop K, Barron KS, Kuehn HS, Hoffmann P, Negro A, Tsai WL, Cowen EW, Pei W, Milner JD, Silvin C, Heller T, Chin DT, Patronas NJ, Barber JS, Lee CC, Wood GM, Ling A, Kelly SJ, Kleiner DE, Mullikin JC, Ganson NJ, Kong HH, Hambleton S, Candotti F, Quezado MM, Calvo KR, Alao H, Barham BK, Jones A, Meschia JF, Worrall BB, Kasner SE, Rich SS, Goldbach-Mansky R, Abinun M, Chalom E, Gotte AC, Punaro M, Pascual V, Verbsky JW, Torgerson TR, Singer NG, Gershon TR, Ozen S, Karadag O, Fleisher TA, Remmers EF, Burgess SM, Moir SL, Gadina M, Sood R, Hershfield MS, Boehm M, Kastner DL, Aksentijevich I. Early-onset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med. 2014 Mar 6; 370(10):911-20.
  2. Zavialov AV, Engstrom A. Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity. Biochem J. 2005;391(Pt 1):51–57. doi: 10.1042/BJ20050683.
  3. Zavialov AV, Yu X, Spillmann D, Lauvau G, Zavialov AV. Structural basis for the growth factor activity of human adenosine deaminase ADA2. J Biol Chem. 2010;285(16):12367–12377. doi: 10.1074/jbc.M109.083527.
  4. Iwaki-Egawa S, Yamamoto T, Watanabe Y. Human plasma adenosine deaminase 2 is secreted by activated monocytes. Biol Chem. 2006;387(3):319–321. doi: 10.1515/BC.2006.042.

Other Diseases with Unknown Mechanism

Degos disease is a poorly understood rare genetic disorder leading to defects in coagulation, in which small veins and arteries become occluded. Initial symptoms present as skin lesions for both the benign and systemic forms of the disease. However, benign Degos is limited to skin lesions, while systemic Degos progresses to other organ systems (intestines, CNS).

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy) is caused by mutations in NOTCH3 and is of slow onset (initial clinical manifestations in the third and fourth decade of life) but progressive with no available cure. Predominant clinical features include strokes, early dementia, migraines, memory loss and multiple psychiatric symptoms.

Premature Coronary Artery disease (premature CAD). Ischemic heart disease is the most common cause of death worldwide, comprising nearly 16 percent of all deaths annual. While many risk factors are known — aging, smoking, diabetes, hypertension, poor diet, lack of exercise — in some cases, the etiology remains a mystery. Is worrisome the increasing recognition of the disease in younger populations, with injury to the inner layer of a coronary artery sometimes manifesting itself as early as childhood, seemingly due to genetic causes. We are continuously working to characterize the etiology and natural history of CADASIL, Degos and premature CAD through comprehensive clinical and molecular evaluations of patients as well as to develop patient-derived disease models to better understand the pathophysiology of these conditions.