We would like to thank Dr. Soshana Svendsen for critical editing. This work was supported by the Israel Science Foundation grant 1621/18, the Ministry of Science and Technology (MOST), Israel 3-15647, the California Institute for Regenerative Medicine grant ID DISC1-08800, the Sherman Family Foundation, NIH-NINDS grant 1UG3NS105703, and The ALS Association grant 18-SI-389. AH was funded by Wallenberg Foundation (grant number 2015.0178).

<ol>
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Cedars-Sinai owns a minority stock interest in Emulate, the company that produces the study&#39;s microfluidic Organ chips. An officer of Cedars-Sinai also serves on Emulate&#39;s Board of Directors. Emulate provided no financial support for this research. Cedars-Sinai and Emulate have patents filed related to this work.

The combination of organ-on-chip technology and iPSC-derived cells in the NVU holds promise for accurate modeling of the human BBB. Here, we provide a detailed protocol for simple and robust application of the recently published iPSC-based BBB chip16. An overview and timing of the seeding paradigm is shown in Figure 3. To obtain and maintain barrier functions that are suitable for BBB modeling, generating a homogenous iBMEC monolayer and retaining its integrity are critical. The first step towards the generation of a functional monolayer includes chemical activation of the nonpolar PDMS surface that allows attachment of ECM proteins. The surface activation reagents degrade rapidly upon reconstitution, which may eventually result in suboptimal attachment of cells. It is therefore important to keep the reagents fresh and protected from light throughout the process.
During UV activation (section 5.2), reagents must be evenly distributed within each channel in order to achieve consistent ECM coating and homogenous cell attachment. ECM compositions and concentrations that are provided in this protocol were optimized to the specific types of cells (i.e., iBMECs and EZ-sphere-derived neural progenitor cells). Changing cell composition is possible but may require differential ECM conditions, which will require optimization. Following ECM coating, it is crucial to seed iBMECs properly. High cell seeding density (&#62;14 x 106 cells/mL) is essential for obtaining a complete monolayer. If failing to achieve full cell confluency, it is advised to further increase the density of the cell suspension up to 20 x 106 cells/mL during seeding.
Laminar flow was previously suggested to enhance iBMEC maturation15 and is indispensable for fulfilling the advantages provided by the microfluidic platform. However, microbubbles flowing through the channels of the chip can physically stress and detach resident cells, which can lead to destruction of the BBB chip integrity. To avoid microbubble formation during laminar flow, it is critical to equilibrate the medium before perfusion is initiated. Equilibration requires pre-warming and degassing of the medium prior to use.
The generation of personalized chips is made possible using both human iPSC-derived iBMECs and iNPCs. While the differentiation of iBMECs is short and rather simple24,22, differentiation of iPSCs into neural cells21,25 is more challenging. However, the neural cells that reside in the &#34;brain side&#34; of the chip can be replaced by any neural cell type, such as primary neural cells or iPSC-derived motor neurons16. Similarly to &#34;bona fide&#34; endothelial cells, iBMECs display plasticity in gene expression in response to different neural co-cultures. Thus, this flexibility may result in future development of various NVUs on the chip. Additional flexibility of the system can be obtained by adjusting the cell seeding timing and order. iBMECs can be seeded prior to or after the neural cells, making it possible to seed iBMECs immediately following seeding and attachment of neural cells, or after EZ-spheres have differentiated into more mature neural cultures in the chip environment. This is of particular importance, given the immature nature of iPSC-derived neural cells. Given that EZ-spheres are early neural progenitors, longer differentiation periods may also result in an increased percentage of astrocytes and neurons.
One component that is missing from the protocol is perivascular pericytes, which provide a crucial component in the physiological NVU. Recent advancements in iPSCs differentiation into pericyte-like cells26,27 will permit the introduction of this additional cell type, thereby providing an improved replica of the BBB while preserving its personalized nature.
IBMECs have previously been shown to demonstrate various properties that are critical for BBB modeling including expression of cellular markers, the establishment of TEER and functional activity of efflux pumps28,24,5. However, molecular analyses have revealed that these cells also express several epithelial markers15. Advancements in iBMEC generation showing improved function and endothelial marker expression have recently been described29,30. Following the paradigm presented here, these potentially improved cells can easily be incorporated into the BBB chip model for future applications. The flexible yet robust platform described here may facilitate both disease modeling and the development and assessment of new CNS drugs.
While this protocol relies on a specific commercially available product, additional commercial companies offer diverse microphysiological platforms, which may offer alternative advantages31. In addition, protocols for &#34;in house&#34; manufacturing of microfluidic Organ chips are also available32 and may offer more modularity including the integration of TEER electrodes17, which is missing from the platform used here.
Organ-on-chips were introduced as an alternative approach to improve the physiological context of current cell cultures33. However, the application of this technology requires specialized engineering skills, which are often lacking in biologically oriented labs. A commercially available chip platform, such as the one employed here, provides less modularity and increased robustness and reproducibility, which can be applied by a wider array of users. Further, the application of laminar flow on a microfluidic chip relies on the application of syringe or peristaltic pumps, which introduces another level of complexity. This obstacle is now easier to overcome with the application of the culture module, which facilitates the simultaneous perfusion of multiple chips.

The BBB is a highly selective barrier that separates the CNS from the circulating blood. It protects critical brain functions from potentially disruptive substances, factors, and xenobiotics while also allowing the influx of nutrients and other metabolites required to maintain brain homeostasis1. The BBB is a multicellular NVU in which pericytes, astrocyte endfeet, and neuronal processes directly contact brain microvascular endothelial cells (BMECs). These interactions allow BMECs to form specialized barrier properties that are supported by tight and adherens junctions2,3. The formation of this barrier limits the paracellular passage of molecules, but it contains polarized transporters to actively transport molecules into the CNS or back into the blood1. Due to these unique barrier properties, the BBB constitutes a major obstacle to the delivery of biopharmaceuticals into the brain, and it is estimated that less than 5% of FDA-approved small molecules can reach the CNS4.
Animal models have been widely used to study BBB penetrance and the molecular mechanisms involved in BBB development5. While animal models faithfully represent the complex multicellular in vivo environment, differences in expression and activity of BBB transporters as well as substrate specificity across species often preclude accurate extrapolation of animal data to humans6. Thus, human-based models are critical to study the human BBB and for use in the development of drugs designed to target the CNS. This need becomes even more apparent with the increasing dominance of biological, human-specific drugs in the pharmaceutical development field. Accumulating evidence suggests that a compromised BBB is associated with a number of severe CNS disorders, including brain tumors and neurological diseases7,8,9. Human models faithfully reflecting these diseases have the potential to both 1) identify novel pathways that could be targeted for drug development and 2) predict CNS penetrance, thus reducing time and resources in preclinical studies and possibly decreasing failure rate in clinical trials.
In vitro models have been widely implemented to study interactions between BMECs and other cells of the NVU and conduct screens for prospective BBB-permeable drugs10. To recreate key aspects of the human BBB, in vitro models must display physiologically relevant properties (i.e., low paracellular permeability and physiologically relevant transendothelial electrical resistance [TEER] across the endothelial monolayer). In addition, the molecular profile of an in vitro system must include expression of representative functional transport systems. Typically, in vitro models are composed of endothelial cells that are co-cultured on a semipermeable membrane with combinations of other NVU cells to enhance BBB properties11. This approach allows simple and relatively rapid assessment of barrier functionality and molecule permeability. Such cell-based BBB models can be established with animal or human cell sources, including cells isolated from surgical excisions or immortalized BMEC lines.
Recently, protocols to differentiate human pluripotent cells into BMECs were introduced as an attractive source for in vitro human BBB models12,13. Induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) are highly scalable, demonstrate crucial morphological and functional characteristics of the human BBB, and carry the genetics of the patient. In culture, iBMECs form a monolayer that expresses tight junction markers and displays in vivo-like tight junction complexes. These cells also express BBB markers, including the BBB glucose transporter, glucose transporter 1 (GLUT1). Importantly, and unlike other alternative cell sources for human BMECs, iBMECs acquire barrier properties with values as high as those measured in vivo14, polarize along the basolateral axis, and express functional efflux pumps. Furthermore, the use of iPSCs from various subjects both 1) welcomes the opportunity to test aspects of the BBB in a personalized medicine manner and 2) provides a flexible source for generating additional cell types of the NVU. Generating these cells from an isogenic cell source to create personalized BBB chips would also aid in understanding inter individual differences in drug responses, which is a major cause for resistance or compromised response to treatment observed in clinical studies.
Use of iBMECs as monolayers in a dish or on a semi permeable transwell insert represents a powerful approach for BBB modeling. These systems tend to be robust, reproducible, and cost-effective. In addition, functional analyses such as TEER and permeability are relatively simple to perform. However, two-dimensional (2D) systems fail to recapitulate the 3D nature of in vivo tissue, and they lack the physiological shear stress forces provided by circulating blood and blood cells. This limits the ability of the vascular endothelium in these models to develop and maintain intrinsic BBB properties and functions.
Microengineered systems lined by living cells have been implemented to model various organ functionalities in a concept called organ-on-chips. By recreating in vivo-like multicellular architecture, tissue-tissue interfaces, physicochemical microenvironments, and vascular perfusion, these microengineered platforms generate levels of tissue and organ functionality not possible with conventional 2D culture systems. They also enable high resolution, real-time imaging, and analysis of biochemical, genetic, and metabolic profiles similar to living cells in the in vivo tissue and organ context. However, a particular challenge of the organ-on-chip is that the design, fabrication, and application of these microengineered chips requires specialized engineering expertise that is usually lacking in biologically oriented academic labs.
We have recently combined iPSC and organ-on-chip technologies to generate a personalized BBB chip model15,16. In order to overcome the technological challenges described, the commercially available Chip-S1 is used together with the culture module, an instrument designed to automate the maintenance of the chips in a simple and robust manner (Emulate Inc.). The BBB chip recreates interactions between neural and endothelial cells and achieves physiologically relevant TEER values, which is measured by custom made organ chips with integrated gold electrodes17. Additionally, the BBB chip displays low paracellular permeability, responds to inflammatory cues at the organ level, expresses active efflux pumps, and exhibits predictive transport of soluble biomarkers and biopharmaceuticals. Notably, BBB chips generated from several individuals captures the expected functional differences between healthy individuals and patients with neurological diseases15.
The protocol detailed below describes a reliable, efficient, and reproducible method for the generation of human iPSC-based BBB chips under dynamic flow conditions. Guidance is provided on the type of assays and endpoint analyses that can be performed directly in the BBB chip or from sampling effluent. Thus, the protocol demonstrates the spectrum of techniques that can be applied for evaluating biological and functional properties and responses in a human-relevant model.
A brief description of the iPSC-based BBB chip is provided here. Human iPSCs are initially differentiated and propagated in tissue culture flasks as free-floating aggregates of neural progenitors, termed EZ-spheres. The top channel of the Chip-S116,18,19 is seeded with dissociated EZ-spheres that form the &#34;brain side&#34; of the chip, as cells differentiate over 7 days into a mixed culture of neural progenitor cells (iNPCs), iAstrocytes, and iNeurons. Human iPSCs are also differentiated in tissue culture plates into iBMECs. The bottom channel of the chip is seeded with iBMECs to form the &#34;blood side&#34; as they develop to form an endothelial tube (Figure 1). The porous extracellular matrix (ECM)-coated membrane that separates the top and bottom channels 1) permits the formation of cell-to-cell interactions between channels and 2) allows the user to run permeability assays and image cells in either channel using a conventional light microscope.

<table><tbody><tr><td>Accutase</td><td>EMD Millipore</td><td>SCR005</td><td>Dissociation solution</td></tr><tr><td>B27</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Bfgf</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Chip-S1</td><td>Emulate Inc</td><td>Chip-S1</td><td>Organ-Chip</td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C5533</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>Dextran-FITC</td><td>Sigma</td><td>46944</td><td></td></tr><tr><td>DMEM: F12</td><td>Thermo Fisher Scientific</td><td>31330038</td><td></td></tr><tr><td>Donkey serum</td><td>Sigma</td><td>D9663</td><td></td></tr><tr><td>Emulate Reagent 1 (ER-1)</td><td>Emulate Inc</td><td>ER-1</td><td></td></tr><tr><td>Emulate Reagent 2 (ER-2)</td><td>Emulate Inc</td><td>ER-2</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Glial Fibrillary Acidic Protein (GFAP)</td><td>Dako</td><td>Z0334</td><td></td></tr><tr><td>GLUT-1</td><td>Invitrogen</td><td>MA5-11315</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050038</td><td>Glutamine supplement</td></tr><tr><td>hBDNF</td><td>Peprotech</td><td>450-02</td><td></td></tr><tr><td>KOSR</td><td>Thermo Fisher Scientific</td><td>10828028</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td>Basement membrane matrix</td></tr><tr><td>mTeSR1</td><td>StemCell Technologies, Inc.</td><td>85851</td><td></td></tr><tr><td>NEAA</td><td>Biological industries</td><td>01-340-1B</td><td></td></tr><tr><td>Nestin</td><td>Millipore</td><td>MAB353</td><td></td></tr><tr><td>NutriStem</td><td>Biological industries</td><td>05-100-1A</td><td>Alternate media</td></tr><tr><td>PECAM-1</td><td>Thermo Fisher Scientific</td><td>10333</td><td></td></tr><tr><td>Platelet-poor plasma-derived bovine serum (PPP)</td><td>Biomedical Technologies</td><td>J64483AB</td><td></td></tr><tr><td>Retinoic acid (RA)</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>S100&amp;beta;</td><td>Abcam</td><td>ab6602</td><td></td></tr><tr><td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td><td>Millipore</td><td>SCGP00525</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>ZO-1 Monoclonal Antibody</td><td>Invitrogen</td><td>33-9100</td><td></td></tr><tr><td>&amp;beta;III-tubulin (Tuj1&amp;alpha;)</td><td>Sigma</td><td>T8660</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Life Technologies</td><td>31350010</td><td></td></tr></tbody></table>

generation of a human ipsc-based blood-brain barrier chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

Figure 6A,B,C represents a BBB chip seeded with EZ-spheres on the &#34;brain side&#34; top channel and iBMECs on the &#34;blood side&#39;&#34; bottom channel. iBMECs were seeded first and allowed to attach overnight, after which EZ-spheres were seeded. Chips were then cultured under static conditions with daily media replacement for seven days. The BBB chip was then fixed using 4% PFA at RT for 10 min and washed 3x with DPBS. Immunocytochemistry was performed on the BBB chip using 1) nestin as a marker for neural progenitor cells, 2) S100&#946; or GFAP as markers for astrocytes, and 3) &#946;III-tubulin as a marker for neurons. GLUT-1 and Pecam-1 were used as a marker for BMECs. Imaging was performed at 20x using a confocal microscope and images were processed using Fiji for ImageJ software.
Figure 6D represents a paracellular permeability assay that was performed on organ chips populated with iBMECs and EZ-spheres, iBMECs alone (without EZ-spheres), or EZ-spheres alone (without iBMECs). Following a &#34;Regulate&#34; cycle, a 4 kDa Dextran-FITC tracer was added to the reservoir of the &#34;blood side&#34; to a final concentration of 10 &#181;g/mL, and chips were perfused at 30 &#181;L/h overnight. Next, media was collected from inlet and outlet reservoirs of both the top and bottom channels. 100 &#181;L of each sample was collected and examined for fluorescence using a plate reader.
Importantly, a 1:1 calibration curve was used to transform fluorescence values into concentration values of Dextran FITC. Values were then used to calculate permeability (Papp). These results demonstrate that iBMECs form functional barrier properties, which are further tightened when iBMECs are co-cultured with EZ-spheres. Chips cultured with EZ-spheres only, fail to form a barrier. A similar approach can be used to examine the transport of any molecule across the BBB chip, depending on an available measurement method (e.g., fluorescence, ELISA, or mass spectrophotometry).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60925/60925fig1a.jpg" /> 
Figure 1: Schematic of the iPSC-based BBB chip. Prior to seeding on the chip, iPSCs are differentiated in culture plates into (i) EZ-spheres (neural progenitor cells, iNPCs), which are grown in suspension as spheres, and into (ii) brain microvascular endothelial cells (iBMECs). EZ-spheres are dissociated into single cells, seeded on the top channel of the organ chip, where they further differentiate into mixed neural cultures to form the &#34;brain side&#34;. iBMECs are seeded in the bottom channel of the organ chip to form a blood vessel-like structure on the &#34;blood side&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60925/60925fig2a.jpg" /> 
Figure 2: Schematic of the top view of the chip in the chip carrier, with labeled ports. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60925/60925fig03.jpg" /> 
Figure 3: Flowchart of the iPSC-based BBB chip preparation and workflow. Pre-differentiation of both neural and endothelial cells from iPSCs is required before initiation of the workflow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60925/60925fig04.jpg" /> 
Figure 4: Seeding of iBMECs in the bottom channel. Brightfield images of iBMECs seeded in the bottom channel (A) immediately after seeding or (B) 24 h post-seeding, after cells have attached. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60925/60925fig5a.jpg" /> 
Figure 5: Schematic of the connected chip and portable module with labeled inlet and outlet media reservoirs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60925/60925fig06.jpg" /> 
Figure 6: Representative results. Immunocytochemistry on the iPSC-based BBB chip 7 days post-seeding. EZ-spheres differentiated into a mixed neural cell population in the top &#34;brain side&#34; channel, including (A) S100&#946;+ (green) astrocytes, Nestin+ (red) neural progenitor cells as well as (B) GFAP+ (red) astrocytes and &#946;III-tubulin+ (red) neurons. Scale bars = 200 &#181;m. (C) iBMECs seeded in the bottom &#34;blood side&#34; channel expressed GLUT-1 and (green) PECAM-1 (CD31, red). Scale bar = 200 &#181;m. (D) Evaluation of BBB chip permeability was performed by adding dextran-FITC (4 kDa) into the reservoir of the bottom channel. Results demonstrate that organ chips seeded with iBMECs and EZ-spheres display a tight barrier compared to organ chips seeded with iBMECs alone (*p &#60; 0.05). Organ chips seeded with EZ-spheres alone do not display any barrier properties (***p &#60; 0.001; one-way ANOVA with Tukey&#39;s multiple comparisons test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60925/60925fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

generation-of-a-human-ipsc-based-blood-brain-barrier-chip

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K Views.  Ben-Gurion University of the Negev. Dit protocol zal aantonen hoe gedifferentieerde geïnduceerde pluripotente stamcellen kunnen worden gezaaid op een orgaan-on-chip om een volledig menselijke, gepersonaliseerde, microfluïde bloed-hersenbarrière te genereren, die kan worden gebruikt om de doorlaatbaarheid van het centrale zenuwstelsel te voorspellen en neurologische aandoeningen te bestuderen. Met behulp van een commercieel verkrijgbaar orgaan-op-chip, zullen we laten zien hoe elk biologisch georiënteerd lab deze technologie...

Video: Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Neuroscience

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Biology

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Bioengineering

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

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