Dr. Valeria Orlova : Pluripotent stem cells for vascular differentiation and disease modeling

Principal Investigator:  Dr. Valeria V. Orlova
Researcher scientists: Oleh Halaidych, Xu Cao, Mees de Graaf, Dr. Gopi Yakala, Dr. Amy Cochraine
Research technician:  Lisa E. van den Hil






Blood vessels are essential for proper functioning of tissues and organs in our body, and vascular dysfunction is associated with many cardiovascular and neurodegenerative diseases. Insight into the  interactions between vascular cells and the local microenvironment are therefore key to understanding many different diseases.  Human induced pluripotent stem cells (hiPSCs) derived from patients with genetic vascular disorders can form blood vessel cells and provide human models for disease that can be used to find new therapeutics and strategies for disease prevention.


Figure 1:2D co-culture of hiPSC-derived ECs and pericytes.

hiPSC-ECs are visualized with CD31 (red), SOX17 (gray) and pericytes are co-stained with SM22 (green); only pericytes close to EC sprouts upregulate SM22; nuclei stained with DAPI (blue). Images taken with EVOS FL AUTO2 system (ThermoFisher), 10X Objective 4X4 focus planes.


Figure 2: 3D microvascular network of hiPSC-ECs and pericytes.

(A) hiPSC-ECs are visualized with CD31 (blue), SOX17 (red), and pericytes are co-stained with SM22 (green); nuclei are stained with DAPI (blue). (B) Confocal image of three-dimensional microvessels formed by hiPSC-ECs (labeled in green): maximum and z-projection are shown. Images are taken with WLL1 Confocal (Leica), 40X Objective.

Aim and focus

We utilize hiPSCs derived from healthy donors and patients with genetic vascular disorders. Over recent years, we have developed efficient protocols to differentiate different endothelial cell (EC) subtypes, pericytes, vascular smooth muscle cells, as well as myeloid cells, such as monocytes and macrophages. Using CRISPR/Cas9 based gene targeting, we generated several fluorescent reporter hiPSC lines for core transcriptional factors (TF) that are being used to advance our understanding of lineage specification and differentiation of ECs with different mesodermal origins and distinguishable by their core transcriptional signature. Specifically we investigate the generation of tissue-specific vascular cells with focus on the heart and brain vasculature. In addition, we are developing vascular cell functionality assays that assess EC barrier function, endothelia-leukocyte interaction under flow (for modelling of inflammation) and Ca2+ and contraction assays for pericytes/vascular smooth muscle cells(vSMCs). In addition, we also use assays for modelling of vasculature and endothelial-pericyte/vSMC interactions in 2D, and in 3D using organ-on-a-chip technology. 

We thus aim to develop realistic disease models for hereditary hemorrhagic telangiectasia (HHT) and cerebral amyloid angiopathy (CAA), and model tumour angiogenesis and inflammation with patient-specific hiPSCs.


Figure 3: Barrier function of hiPSC-ECs. (A) Adherens junctions (Ve-cadherin, in red) and actin (Phalloidin, in green) in hiPSC-ECs. (B) Disassembly of adherens junctions (Ve-cadherin, in red) and actin (Phalloidin, in green) in hiPSC-ECs induced by Thrombin (0.1 U/ml). (C) Real-time measurements of barrier breakdown using electric cell-substrate impedance sensing (ECIS) system (Applied Biophysics).


Figure 4: Gene networks of hiPSC-ECs derived from different mesoderm subtypes. 


Collaboration within the LUMC

Dr. Franck Lebrin (NIER) who carries out complimentary studies on HHT in transgenic mouse models.
 
Prof. Judith Bovee (PATH) on hiPSC model of vascular tumors, i.e. pseudomyogenic (epithelioid sarcoma-like) hemangioendothelioma. 

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Prof. Peter ten Dijke (CCB) on hiPSCs to Model Human Fibrodysplasia Ossificans Progressiva (FOP).
 
Prof. Anton Rabelink (NIER) on hiPSC derived endothelial cells.


External Collaboration

Dr. Arie Reijerkerk (Ncardia) on scaled production of hiPSC endothelial cells in bioreactors. 

Dr. Andries van der Meer (University of Twente) on hiPSC-derived endothelial cells and age-related macular degeneration (AMD). 

Dr. Roxanne Kieltyka (Leiden University) on hiPSCs and supramolecular biomaterials. 

Prof. Konstantinos Anastassiadis (BIOTECH TU Dresden) on hiPSCs and genetic fluorescent reporters.


Grants and other funding

Netherlands Organ on Chip Initiative – NOCI (NWO – Netherlands Organization for Scientific Research), 2017.

Tools and TECHNOlogies for Breakthrough in hEArt Therapies – TECHNOBEAT (Horizon 2020), 2016.

Understanding and treating vascular disorders: research at the roots of degenerative disease and tissue repair (Möller Foundation Research Grant, The Netherlands), 2014.

Pluripotent stem cell resources for mesodermal medicine – PluriMes (EU FP7), 2014.

Vascular derivatives from patient specific induced pluripotent stem cells (iPSCs): a novel platform for mechanism specific drug discovery for hereditary haemorrhagic telangiectasia (HHT)
(SWORO – Dutch HHT Foundation), 2013.

Gisela Thier Fellowship (LUMC, the Netherlands), 2010.

Key Publications

1.        Giacomelli, E., Bellin, M., Sala, L., van Meer, B. J., Tertoolen, L. G. J., Orlova, V. V., and Mummery, C. L. (2017) Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells. Development. 10.1242/dev.143438

2.        Passier, R., Orlova, V., and Mummery, C. (2016) Complex Tissue and Disease Modeling using hiPSCs. Cell Stem Cell.18, 309–321

3.        Cai, J., Orlova, V. V., Cai, X., Eekhoff, E. M. W., Zhang, K., Pei, D., Pan, G., Mummery, C. L., and Dijke, ten, P. (2015) Induced Pluripotent Stem Cells to Model Human Fibrodysplasia Ossificans Progressiva. STEMCR. 5, 963–970

4.        Birket, M. J., Ribeiro, M. C., Verkerk, A. O., Ward, D., Leitoguinho, A. R., Hartogh, den, S. C., Orlova, V. V., Devalla, H. D., Schwach, V., Bellin, M., Passier, R., and Mummery, C. L. (2015) Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nature Biotechnology.33, 970–979

5.        Orlova, V. V., Drabsch, Y., Freund, C., Petrus-Reurer, S., van den Hil, F. E., Muenthaisong, S., Dijke, P. T., and Mummery, C. L. (2014) Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts. Arteriosclerosis, Thrombosis, and Vascular Biology.34, 177–186

6.        Orlova, V. V., van den Hil, F. E., Petrus-Reurer, S., Drabsch, Y., Dijke, ten, P., and Mummery, C. L. (2014) Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nature Protocols.9, 1514–1531

7.        van der Meer, A. D., Orlova, V. V., Dijke, ten, P., van den Berg, A., and Mummery, C. L. (2013) Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab Chip.13, 3562–3568

8.        Economopoulou, M., Langer, H. F., Celeste, A., Orlova, V. V., Choi, E. Y., Ma, M., Vassilopoulos, A., Callen, E., Deng, C., Bassing, C. H., Boehm, M., Nussenzweig, A., and Chavakis, T. (2009) Histone H2AX is integral to hypoxia-driven neovascularization. Nature Medicine.15, 553–558

9.        Orlova, V. V., Choi, E. Y., Xie, C., Chavakis, E., Bierhaus, A., Ihanus, E., Ballantyne, C. M., Gahmberg, C. G., Bianchi, M. E., Nawroth, P. P., and Chavakis, T. (2007) A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin. The EMBO Journal.26, 1129–1139

10.      Orlova, V. V., Economopoulou, M., Lupu, F., Santoso, S., and Chavakis, T. (2006) Junctional adhesion molecule-C regulates vascular endothelial permeability by modulating VE-cadherin-mediated cell-cell contacts. Journal of Experimental Medicine.203, 2703–2714


All Publication list can be found here:

https://www.ncbi.nlm.nih.gov/sites/myncbi/1hURjjjrveoQr/bibliography/54475445/public/?sort=date&direction=ascending