Cover Page
The handle http://hdl.handle.net/1887/80758 holds various files of this Leiden University
dissertation.
Author: Halaidych, O.V.
Title: Towards functional analysis of cerebrovascular cell types derived from human
induced pluripotent stem cells
Chapter 6
DISCUSSION
The network of cerebral blood vessels in the brain ensures the supply of oxygen and nutrients and removal of carbon dioxide and cerebral metabolites in the Central Nervous System. Malfunction leads to many different diseases including cognitive dysfunction and dementia. Understanding the role of the vasculature in physiological and pathophysiological conditions requires representative and reliable experimental models. Multiple in vivo and ex vivo animal models have provided great tools for studying vasculature in normal and diseased conditions and have provided a wealth of information on genetic diseases that affect the vasculature. However, there are crucial differences inphysiology, metabolism, immune system, inflammation, and genetic backgrounds with humans that put important limitations on using animal models as translational tools for studying human vascular diseases and drug development. Ex vivo models based on primary human tissue and cells are also of great importance but given its limited availability, donor-to-donor variability, genetic background effects and difficulties in maintaining phenotypes in culture they cannot provide a renewable experimental model. Immortalized human cell lines are also widely used but they often fail to reproduce the features of native tissue cells. Cellular models that are both immortal, are representative of real blood vessels, can be standardized and show higher reproducibility are greatly needed. The work described in this thesis contributes to these outstanding challenges by developing models based on human induced pluripotent stem cells to create mimics of vasculature in the human brain.
Human induced pluripotent stem cells (hiPSCs)
assessment of hiPSC-derived vascular cellular components with emphasis on cerebral vasculature.
hiPSC-derived endothelial cells
Discovery of hiPSC which can differentiate into functional vascular cell types served as a strong driver of contemporary biology and vascular research particularly [1]. Commonly used endothelial cell (EC) differentiation protocols are based on 2D monolayer protocols (discussed in Chapter 1). Successful differentiation of ECs is at present best achieved by mimicking embryonic developmental. First, hiPSCs are directed toward the mesoderm lineage. At this stage, mesoderm cells may adopt a paraxial, intermediate or lateral plate fate. In Chapter 2 and 3 we used a lateral plate mesoderm differentiation conditions followed by vascular specification using VEGF [12,13]. Although one of our aims was to examine brain endothelial barrier function, these differentiated ECs did not have BBB-like characteristics, as expected given their developmental history. Several studies have reported on induction of brain microvascular ECs from hiPSCs [14–17]. However, it remains to be confirmed whether these cells actually represent true brain ECs and how long they can retain a BBB-like phenotype in
vitro. Considerable work is also still required to make other types of vascular
cells like arterial-, capillary- and venous-specific ECs to allow precise mimicking of different vascular beds. This thesis contributed to characterization and establishing the standardized assessment of barrier function, wound healing, formation of vascular plexus and inflammatory responses of hiPSC-derived ECs.
In Chapter 2 we performed a side-by-side functional comparison of hiPSC-ECs and primary human hiPSC-ECs. hiPSC-hiPSC-ECs were derived from two independent hiPSC lines generated using a Sendai virus (SeV)-based (non-integrating/ DNA-free) reprograming method [18,19] and isolated using two independent “magnetic bead-based” methods: CD34+ cells on day 6 of differentiation and CD31+ cells on day 10. We chose human dermal blood ECs (HDMECs, isolated from one donor) and human umbilical vein endothelial cells (HUVECs, isolated from 1) two independent donors and 2) two independent batches for one of the donors) as the sources of human primary ECs.
Firstly, we performed a comparative assessment of barrier function and real-time migration using electrical cell-substrate impedance sensing (ECIS). Examination of barrier function revealed that CD31+ hiPSC-ECs had tighter barriers than either CD34+ hiPSC-ECs or HUVECs or HDMECs. We also investigated the effect of well-known barrier disrupting agents, histamine and thrombin. We found that hiPSC-ECs showed pronounced responses to relatively low concentrations of thrombin (0.1 U/mL) but they failed to fully recover the barrier in contrast to primary cells. We also found that hiPSC-ECs, like HUVECs, do not respond to Histamine, whilst dermal microvascular ECs (HDMECs) are the only cells that showed a reduction in barrier function upon Histamine stimulation. This could be related to the vascular bed origin of the cells and their specific physiology to this type of response.
capacitive component (C) of impedance at high frequencies as a measure of confluence of the endothelial monolayer, allowed us to quantify cell migration rate more precisely using the first derivative (dC/dt) and to estimate the total time of monolayer recovery with high reproducibility. We found that hiPSC-ECs were highly sensitive to VEGF. Although not observed in primary hiPSC-ECs, hiPSC-ECs demonstrated both a pronounced decrease in migration rate as well as in the barrier after serum-free conditioning. Taken together, ECIS-based assessment of hiPSC-ECs proved to provide an informative and reproducible functional characterization and quality control assay and allowed the cells to be compared directly with primary ECs. Importantly, ECIS assays provided a means of quantitative assessment of EC behavior using mathematical tools for data analysis. This was the main outcome of this part of the study.
In Chapter 2 we also investigated the ability of hiPSC-ECs to form a two-dimensional vascular plexus and compared this with primary human ECs. To achieve this, we used an in vitro assay in which hiPSC-ECs were co-cultured with different sources of stromal cells [13]. We observed that hiPSC-ECs were more sensitive to the source of stromal cells compared to primary ECs. hiPSC-ECs were able to form very dense vascular networks with high total vessel length and number of junctions. It is noteworthy that selecting the right stromal cell source is important. For this reason, the assay potentially can be used to test whether hiPSC-derived SMCs or pericytes of interest can support endothelial sprouting of control ECs, either primary or hiPSC-derived. To take this a step further, we showed that hiPSC-ECs were able to form lumenized vessels in vivo. When hiPSC-ECs were co-transplanted with primary human bone marrow stromal cells (BMSCs) as Matrigel plugs into mice, they formed perfused vascular networks, as indicated indirectly by the presence of red blood cells inside the vessels in immunohistochemistry sections.
Interaction of ECs with immune cells is an important aspect of disease. Comparison of inflammatory responses revealed that hiPSC-ECs reacted to pro-inflammatory stimulation by tumor necrosis factor alpha (TNFα) in a similar but not identical manner as HUVECs. Although we observed upregulation pro-inflammatory adhesive receptor E-selectin and ICAM-1 similar to HUVECs, hiPSC-ECs showed no upregulation of VCAM-1 in contrast to HUVECs. This is in line with previous reports, as primary ECs were also shown to exhibit differential upregulation of VCAM-1, much like ECs from different organs such as different compartments of the kidney vasculature, where there is prominent VCAM-1 expression in arteriolar endothelium but not in glomerular endothelium [20,21]. Examination of adhesion of human leukocytes to ECs under physiological flow revealed that hiPSC-ECs pre-treated with TNFα were capable of capturing leukocytes, although with lower efficiency than primary HUVECs.
Although HUVECs are still often used in preference to hiPSC-ECs in in vitro assays, our comprehensive characterization and line-to-line and batch-to-batch comparisons of hiPSC-ECs provided a strong argument for using hiPSC-ECs instead of primary ECs. Overall consistency between different (healthy) hiPSC-EC lines suggested that hiPSC-hiPSC-ECs can even be considered as a benchmark for standardization of functionality across different lines.
successful dissociation of ECs and seeding into microfluidic channels, (3) step-by-step preparation of microfluidic setup for precise control of fluid flow and perfusion of leucocytes labeled with fluorescent tracer, (4) the live imaging setup and (5) description of all critical steps for correct automated counting of adherent leucocytes using freely available image processing software. The protocol allowed elucidation of the interaction of ECs and leukocytes but did not take into account other cell types, such as vSMCs or pericytes which are also present in the vascular wall and might have an influence on leukocyte extravasation. Additionally, by capturing image sequences instead of end-point images and applying automated cell tracking software this assay potentially can be used to examine the whole cascade of endothelial-leukocyte interactions, including anchoring and rolling. Despite these limitations, the flow assay described for the assessment of inflammatory responses provides a valuable tool for basic characterization and disease modeling applications of hiPSC-ECs.
hiPSC-derived mural cells
Blood vessels are not only made up of ECs but also mural cells (vSMCs and pericytes) of various identities that surround the EC tubular structures. Given that the interaction with these cells can affect EC behavior, this thesis also aimed to provide a robust approach for quantification and characterization of vSMCs and pericyte functionality in vitro.
Mural cells are non-striated muscle cells that have the ability to contract upon release of Ca2+ from intracellular stores. Changes of intracellular Ca2+ levels take a
variety of forms, ranging from temporally varying subcellular (local) to cell-wide (global) changes. The latter are correlated with contractile states of perivascular cells thus enabling control of blood vessel lumen and therefore blood flow. Because of significant heterogeneity of perivascular cell phenotypes (mentioned in Chapter 1) assessment of contractile responses and Ca2+ signaling in these cells
is complex. In Chapter 4 we tested and implemented a computational method for characterization of (1) global intracellular Ca2+ release upon controlled addition
of a vasoconstrictor using a precisely-controlled microfluidic pump and (2) cell contraction in single cells at the population level. Examination of large ensembles of human brain vascular pericytes (HBVPs) and smooth muscle cells (HBVSMCs) revealed dispersion of Ca2+ release and contraction parameters that may normally
be overlooked using conventional manual methods. Importantly, qualitative differences in the number of Ca2+ events within single cells suggested a functional
of cells attached to stiff surface reflects isometric contraction, it provides a much more standardized assay that can be easily reproduced in many biological laboratories without the need for specialized soft microengineering facilities and complex image processing and data analysis.
The two assays we developed overcame issues of traditional (time-consuming) manual analysis methods which are prone to biased selection of prominent “eye-catching” cells (e.g. highly oscillating cells, cells with notably decreased surface area, etc) often resulting in collection of statistically unrepresentative samples that do not reflect the heterogeneity of parameters in the whole cellular population. On this evidence, quantitative comparison of large samples with commonly used statistical tests turns out not to be informative as it results in statistical significance with extremely low p-values even upon small deflections of the absolute value observable. Moreover, such tests give only binary answers to the question of whether two samples compared are different or not. In Chapter 4 we, therefore, applied a discrete estimator of Kullback-Leibler divergence to quantify the extent of divergence of two populations which provided the answer of how different two compared populations are.
Over the duration of this project, significant progress has been made by ourselves and others in differentiation of hiPSCs into vSMCs and pericytes. As discussed in Chapter 1, broad heterogeneity intrinsic to these cells is first and foremost due to (1) their diverse embryological origins, (2) their ability to undergo phenotype switching and (3) their location in different vascular beds where local cells can affect their identity. This causes complications in achieving a true in
vivo phenotypic mimic. The derivation of tissue-specific perivascular cells is thus
challenging and at present relies on deep knowledge of their developmental origin. In Chapter 5, the aim was to derive cerebral vSMCs. hiPSCs were directed towards the neuroepithelial lineage to derive a neural crest cell population. Using a protocol adapted from previous work [22,23] we obtained robust induction of neural crest cells (NCCs) in defined culture medium. Notably, NCCs could be easily expanded and cryopreserved increasing reproducibility of subsequent differentiation toward vSMCs for functional assays. Comparison of differentiated NC-SMCs to primary HBVPs and HBVSMCs (all cells of neural crest origin) allowed us to confirm their identity.
HBVPs and HBVSMCs using a method described in Chapter 4. Pairwise analysis of differences identified NC-SMCs that exhibited Ca2+ release with reoccurring
events (oscillations) similar to that observed in HBVSMCs and showed highest values of cell contraction and those NC-SMCs that had predominantly only one Ca2+ release as observed in HBVPs and showed low cell contractility. In general,
hiPSC-derived NC-SMCs showed slower Ca2+ release and reuptake that might be
present due to their immature phenotype. Having tested NC-SMCs differentiated with two protocols from three independent healthy hiPSC lines we observed high consistency of results. Overall, we expect this methodology to provide a robust approach for quantification and specification of vSMC functionality with high reproducibility.
Future perspectives
Healthy vasculature relies on highly orchestrated interaction of heterotypic cellular components. On the one hand, reductionistic approaches for studying these components independently without interaction between one another can be extremely insightful in that it allows elucidation of important molecular or cellular aspects of interest in complex physiological or pathophysiological vascular processes. On the other hand, it might miss essential interactions between cells that may manifest themselves only upon interplay. The latter can be studied in
vitro in coculture conditions where ECs, pericytes, vSMCs, and cells of local tissue
(for instance, in case of cerebral vasculature these might be astrocytes, neurons, microglia) are cultured together. Taking into account a general observation that hiPSC-derived cell types usually exhibit immature characteristics compared to those of primary cells [24], complex multicellular models may facilitate their mutual maturation.
Moreover, the physical parameters of experimental models play their important roles. Incorporation of biophysical strain and stretch, fluid flow, in
vivo-like 3D topology can potentiate vascular cell maturation. The relatively new
and rapidly growing field of “Organ-on-chip” technology brings a new class of
in vitro models that combine heterotypic cellular cultures and micro-engineered
devices, or “microfluidic chips”, to model processes of tissue- and organ-level physiology [25]. Several organ-on-chip models in which ECs are co-cultured with other cells have been developed ([26–28] (reviewed in [29]), including lung-on-a-chip [30] and BBB-lung-on-a-chip [31–33].Together with hiPSC technology and advances in efficient genome editing tools, this promises a new era in patient-specific disease modeling, drug development and regenerative medicine [34,35]. Added complexity brought by multicellular models incorporating micro-engineered devices in a 3D environment would require adaptation and, in some cases, a substantial revision of available functional assays. For instance, of particular interest would be to study vasomotion of EC-vSMC coculture forming pseudo vessels in 3D. In such a system, vSMCs would cause vasoconstriction and the presence of ECs would potentially allow relaxation via nitric oxide (NO) release as well as propagation of Ca2+ waves. Apparently, loading of coculture
[38]) of distinct emission spectra. Addition of the third, host tissue cell type (e.g. astrocytes, neurons or immune cells) would make the research options even more exciting. Aspects of this thesis contributed to laying the ground work to these kinds of approaches.
Assessment of endothelial barrier and transport function in 3D also presents certain challenges. No doubt, methods based on the passage of labeled tracer molecules will remain a valuable experimental tool. However, impedance spectroscopy proved to be extremely insightful and its adaptation in 3D would be valuable. Future studies should focus on improving the positioning of the electrodes required for precise impedance measurements which largely depend on the exact geometry of biochips and spatial distribution of incorporated cells.
Another important breakthrough of recent years in developing more complex in vitro hPSCs-based 3D culture models to capture tissue reality was the initiation and expansion of organ-like structures, called organoids [39]. Organoids resemble a variety of organs by mimicking their in vivo counterparts with conserved functionality. In particular, the development of human brain organoids is aimed at the generation of diverse brain regions to enable a detailed study of the pathogenesis of inherited and acquired brain diseases [40]. Vascularized brain organoids would be invaluable as part of an in vitro model of the whole neuro-vascular unit and BBB in particular. Human development studies would benefit from the further improvement of organoid models as they have a potential to prevail over commonly used post-mortem and pathological specimens, non-human primates or mouse models. Of note, organoids from adult organs only represent the epithelial component of tissue and not the stromal or vascular compartment. Future studies of organoid protocols will have to overcome many limitations, including low reproducibility, incomplete cell type diversity, slow maturation, and vascularization.
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