Therapeutic development for human diseases continues to face obstacles, particularly in translating targets or compounds identified by in vitro screening campaigns to valid targets or efficacious and safe compounds once tested in humans.  Here we discuss strategies that leverage induced pluripotent stem cells (iPSCs) to increase the relevance of human cell models for these in vitro approaches.  We review current advances in genome engineering to pivot from challenges with delivery, identification and selection and subsequent clonal outgrowth by leveraging tools to consistently and reliably generate knock-out and knock-in models for use in target or compound identification.  Specifically, we demonstrate this approach with iPSCs to build isogenic disease models, which can be further differentiated to various cell types of interest such as cardiomyocytes and dopaminergic neurons to model disease and are more directly related to a disease area than commonly used immortalized cell lines.  We expect strategies combining genome engineering and stem cells to provide platforms for more robust disease models that will provide more predictable translation of in vitro to in vivo results.

Adult stem cell research is one of the most dynamic and fast growing fields of our time. Investigating how organs maintain and repair themselves has led to the discovery of cell populations throughout the body that hold immense regenerative capacity. For a long time, research on adult stem cells has been held back by the inability to grow and study these cells in vitro. This limitation has finally been overcome with the development of organoid technology, a system that enables us to grow single adult stem cells into self-organizing miniature organs. These organoids perform many functions of their parent tissue and are excellent tools to study organ regeneration, organ function and disease development. This presentation provides an overview of the many contributions the team around Hans Clevers has made to the development of organoid culture and delves into applications of the technology in disease modeling, pharmacological screening, regenerative medicine and cancer research.

Derivation of many different cell types from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotent stem cells or hiPS cells) is an area of growing interest both for potential cell therapy and as a platform for drug discovery and toxicity. Most particularly, the recent availability of methods to introduce specific disease mutations into human pluripotent stem cells and/or to derive these cells as hiPS cells by reprogramming from any patient of choice, are creating unprecedented opportunities to create disease models “in a dish” and study ways to treat it or slow down its rate of development. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. We have used these methods to produce isogenic pairs of hiPSC lines to compare diseased and corresponding control cardiomyocytes and vascular endothelial cells and identify disease related phenotypes and mechanisms. The use of isogenic pairs has proved crucial since variability between “healthy control” hiPSC lines is often greater than the difference between a diseased cells and its isogenic control.  We have also examined drug responses of hESC-derived cardiomyocytes to a variety of cardiac and non-cardiac drugs and shown that iPSC derived cardiomyocytes with mutations in ion channel genes can accurately predict changes in cardiac electrical properties and reveal drug sensitivities also observed in patients. Similar studies will be described using vascular endothelial cells from hPSC. Relevant in all cases is the development of appropriate bioassays in which to measure disease phenotypes which may be highly cell type specific dependent. For heart cells, this might be electrical activity or contractions force; for vascular cells, responses to fluid flow flow and inflammation. Various approaches to this will be presented.

Concerted efforts over past decades have established a thorough understanding of the canonical somatic DNA methylation landscape as well as its systematic misregulation across many cancers. However, the underlying mechanisms that may direct this genome-scale transformation remains elusive, with no clear model for its acquisition or understanding of its potential developmental utility. I will first summarize work from the past decade that provided key insights into our general understanding of DNA methylation and then discuss recent data that provide evidence for a developmental origin of the shared, erratic cancer methylome.

Regulatory non coding RNAs, including microRNAs (miRNAs) and long non coding RNAs (lncRNAs), have shown to be essential for animal development and viability, yet dissecting the relevance of individual miRNA or lncRNA has been challenging for a given cell context. In addition, the role of ncRNAs for neurodegeneration is still obscure. To identify ncRNAs participated during motor neuron development and degeneration, we used human embryonic stem cell and motor neuron disease iPSCs as paradigms and robustly harnessed them into different motor neuron subtypes to perform strand specific RNA-seq and small RNA-seq simultaneously. We identified several novel MN signature miRNAs and lncRNAs, and systematically analyzed their functions by generating knockout mice. I will present the current progress of the characterization of these MN-ncRNAs by gain-of-function and loss-of-function studies. Collectively, these results will provide critical information for ncRNAs function and will fill in the information on how ncRNAs mediate MN development in vivo.

Recent FDA approval of the first Chimeric Antigen Receptor T cell (CAR-T) therapy offers cancer patients more promise than ever for curative effects. However, many technical challenges in T cell gene delivery still remain in order for this therapy to become a standard of care practice. In this webinar, we will highlight the different viral and non-viral delivery approaches used in T cell engineering for cell and gene therapy applications including:

New solution for small-to-large scale serum-free, suspension lentiviral production – LV-MAX™ Lentiviral Production System

• Platform development process using Design of Experiment (DOE) methodologies
• High-throughput to large scale bioreactor protocols
• Cost benefits of this system over current methods

Novel gene editing tools for primary T cells

• New potent gene editing tools to increase knock-in and knock-out efficiency
• Addressing non-viral delivery barriers through protocol optimization

Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson’s disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

The human inner ear contains ~75,000 sensory hair cells that detect sound or movement via mechanosensitive hair bundles and transmit signals to the brain via specialized sensory neurons. Inner ear sensory cells derived from pluripotent stem cells would provide a valuable model for drug testing, yet a defined differentiation approach has been challenging to identify. Our group has established a step-wise method for generating inner ear organoids, with functioning hair cells, from human pluripotent stem cells. In a 3D culture system, we modulated TGF, BMP, FGF, and WNT signaling to direct the self-organization of multiple otic vesicle-like cysts from a homogenous cell aggregate. Over a 60-day period, the vesicles developed into multi-chambered organoids with sensory epithelia containing hair cells, reminiscent of the membranous organs of the inner ear. This presentation will discuss the process of making inner ear organoids as well as the potential applications of this technique as a powerful tool for investigating human inner ear development and drug discovery.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) as well as engineered heart muscles offer great potential for regenerative applications by CM transplantation, for the study of cardiac development and disease modeling, as well as for drug discovery and cardiotoxicity screenings in a human physiologically relevant model system. Several optimized protocols are available to efficiently differentiate iPSCs into CMs, which possess structural and functional properties of fetal CMs. However, the current methods produce a heterogeneous population of ventricular, atrial and pacemaker-like CMs strongly limiting their field of application. The generation of homogenous populations of subtype-specific iPSC-CMs and their comprehensive phenotypic comparison is crucial for a better understanding of the predominantly cardiac subtype-restricted disease mechanisms as well as for regenerative and pharmacological applications.

The goals of this study were to develop an efficient method for the directed differentiation of human iPSCs into defined functional CM subtypes in feeder-free culture conditions and to obtain a comprehensive understanding of the molecular, cell biological and functional properties of atrial and ventricular iPSC-CMs on both single cell as well as tissue level.

On the basis of temporal modulation of canonical WNT and retinoid acid signaling throughout differentiation of iPSCs via small molecules, we were able to guide the cardiac progenitor cells towards distinct cell fates resulting in homogeneous populations of either atrial or ventricular CMs. Transcriptome and proteome profiling as well as functional analysis of the CM subtypes via optical action potential screening, calcium imaging as well as engineered heart muscles demonstrate that atrial and ventricular iPSC-CMs highly correspond to CMs from the human atrium and ventricle, respectively.

In summary, this study provides a comprehensive understanding of the molecular and functional characteristics of atrial and ventricular iPSC-CMs and supports the suitability of these cells for its application in more precise disease modeling, drug screening as well as for cell-based therapeutic approaches.

The continuous generation of blood cells throughout life relies on the existence of hematopoietic stem cells (HSC) generated during embryogenesis. Given the importance of HSC transplantation in cell-based therapeutic approaches, considerable efforts have been made toward understanding the developmental origins of embryonic HSC. Adult-type HSC are first generated in the aorta-gonad-mesonephros (AGM) region between days 27 and 40 of human embryonic development. It is relatively well accepted that the HSC emerge in the AGM through a hemogenic endothelium, but the direct precursor of this cell type remains to be clearly identified. The current picture of human hematopoietic development is largely extrapolated from animal models (mouse and zebrafish), as comparable studies on the human embryo are not possible. Human pluripotent stem cells (hPSCs) offer unprecedented opportunity to complement this knowledge, assuming that differentiation in the culture dish can recapitulate the dynamic and complexity of hematopoietic development in vivo. Here we summarize the recent advances made to understand the origins of hematopoietic stem cells in the early embryo and we discuss in detail latest protocols to obtain HSC from hPSCs.

Bioflavonoids are dietary agents found in everyday food items such as fruits, vegetables, and legumes. It has been suggested that bioflavonoids be used with chemotherapeutics as an anti-cancer agent due to their anti-inflammatory capabilities and their ability to inhibit the DNA damage response. However most drugs that inhibit the DNA damage response can have secondary effects on normal cells. For instance, chemotherapeutics, such as etoposide, can cause DNA translocations leading to malignant transformations of normal cells to cancer cells. Studies have shown that etoposides and other chemotherapeutics increase the risk of leukemia. Therefore, it is our hypothesis that bioflavonoids inhibit the DNA damage response in normal cells, leading to improper repair of DNA damage, such as chromosomal translocation that can lead to leukemia. To study this we will use mouse embryonic stem cells, to model both acute high-dose exposure and chronic low-dose exposure to bioflavonoids such as genistein, quercetin, luteolin, myricetin etc. After we expose cells to these bioflavonoids, we will be examining them for DNA damage using Immunohistochemistry (IHC) to identify phosphorylation histone variant 2AX, a well-studied marker of DNA damage, with confocal microscopy. We will also be using a translocation reporter construct, which causes cells to produce green fluorescent protein when a translocation has occurred. We have integrated this construct into a mouse embryonic stem cell line, to measure incidences of DNA translocation by flow cytometry. Finally, we will be collecting DNA from these cells for PCR analysis and DNA sequencing to verify any DNA translocations that have occured.

Therapeutic application of pluripotent stem (PS) cell-derived products represents the ultimate goal of stem cell research. In order to apply this technology to patients, it is fundamental to characterize in detail the cell population of interest and identify strategies for its purification from unwanted cells using clinically-compatible methods. In the case of skeletal muscle wasting disorders, we have shown that human PS cell-derived PAX7-induced myogenic progenitors may represent an excellent candidate for cell therapy. To successfully translate this approach toward the clinic, we took advantage of next-generation sequencing techniques to dissect PAX7 function during human myogenesis. Combination of PAX7 genomic target profiling using ChIP-seq and whole transcriptome analysis (RNA-seq), in which we systematically evaluated different time points of the PAX7-dependent myogenic commitment from human PS cells, revealed a subset of genes differentially expressed at various stages of this differentiation process, including a discrete number of surface markers. After Fluorescence Activated Cell Sorting (FACS)-mediated screening, we identified α9β1 integrin, CD54 and Syndecan2 (SDC2), as potential surface markers to be used for the prospective isolation of human PS cell-derived myogenic progenitors. We demonstrate that these surface molecules reproducibly allow for the isolation of myogenic progenitors from multiple human ES/iPS cell lines, in both serum- and serum-free culture conditions, and that α9β1+CD54+SDC2+ (triple+) cells represent a homogenous population of PAX7+ cells endowed with in vivo muscle regeneration potential. Furthermore, we demonstrate that a single marker is sufficient for the magnetic-based isolation of myogenic progenitors, thus enabling adaptation of our differentiation protocol to cGMP standards. These novel findings provide a clinically relevant method for the purification of PS cell-derived muscle progenitors for clinical applications.

How often are you asked what you do and when you start to explain you see the person you are talking to start to twitch, their eyes glaze over and they are desperately trying not to check their phone for something more interesting. One way to avoid that is to have an “elevator pitch” explanation ready. This is simply a plain English, well-rehearsed explanation about what you do, why it’s important and why people should care. It sounds easy but it’s not. This brief section will explain the key things you need to keep in mind when developing an “elevator pitch” and the things you definitely need to avoid.

Regenerative medicine therapeutics aimed at restoring normal cell function due to trauma and disease are gaining increasing momentum in clinical trials. For this purpose, lentivirus-based gene delivery is actively being investigated to treat a collection of monogenic and neoplastic diseases. The recent success of CAR-T-cell products further indicate an emerging role for lentiviral therapies, however, scalability and cost-effective production still present manufacturing challenges. To circumvent these problems, transient lentivirus production has been proposed in suspension host cells, yet consistent high titer yields remain difficult to achieve. In this presentation, a lentiviral production screen will be discussed where different cell lines, transfection reagents and media have been assessed to define a baseline process for scalable and cost-effective manufacturing of high titer lentivirus.

Human pluripotent stem cells (hPSCs) and their derivatives are increasingly being used in industry to gain a competitive edge in drug discovery and a new wave of biotechnology start-up companies are poised to evaluate hPSC-derived cell therapies.  Over the past decade protocols to a broad repertoire of hPSC-derived cell fates have been identified and published differentiating pluripotent stem cells to generate cells more capable of recapitulating phenotypes seen in primary cells.  Although, commercial uses for these cells are quite similar to academics on the day-to-day, there are stark differences in how industry plans to use pluripotent tissue.  Seen through the lens of industry, approaches to high throughput screening, how one employs disease modeling, and the overall need for greater scale and reproducibility become greater priorities.

Worldwide hPSC-derived cells are being evaluated in clinical trials, and with great enthusiasm within the United States several industry and academic efforts are underway to determine if a cure can be found.  These endeavors further underscore the importance of keeping the technical limitations of hPSCs and their derivatives in mind.  Numerous hurdles still remain before cells can make it to the clinic.  Cells will have to be thoroughly vetted for safety, engineering solutions need to be identified for large-scale production, and protocols require adaptation to adhere to good manufacturing practices.  Even then, limitations remain as to appropriate disease indications for cellular therapies.

Current methods for human induced pluripotent stem cells (hiPSC) derivation, expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are reprogrammed and expanded as cell-MC aggregates and then directly differentiated in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. Here, this presentation describes the principles of using IPS-Spheres™ based integrated hiPSC derivation, expansion and differentiation process. Finally examples of integrated process for generation blood cells as well as challenges to be solved are described. 

There are a number of free and independent scholarly publishing tools available that save journal authors time and ensure research is found, read and tracked. In this talk, we will explore three of them:

• ORCID - a persistent digital identifier that distinguishes you from every other researcher and, through integration in key research workflows such as manuscript and grant submission, supports automated linkages between you and your professional activities ensuring that your work is recognized.
• Digital Science’s Altmetric - thousands of conversations about scholarly content happen online every day. Altmetric tracks a range of sources to capture and collate this activity, helping you to monitor and report on the attention surrounding the work you care about.
• Kudos makes sure more people find, read, apply and cite your research. Kudos provides you with simple tools and guidance to help you maximize readership and citations for your work.

The identity of somatic and pluripotent cells can be epigenetically reprogrammed and forced to adapt a new functional cell state by different methods and distinct combinations of exogenous factors. The aspiration to utilize such ex vivo reprogrammed pluripotent and somatic cells for therapeutic purposes necessitates understanding of the mechanisms of reprogramming and elucidating the extent of equivalence of the in vitro derived cells to their in vivo counterparts. In my presentation, I will present my group’s recent advances toward understanding these fundamental questions and further detail our ongoing efforts to generate developmentally unrestricted human naive pluripotent cells. I will conclude by highlighting new avenues for utilizing epigenetic reprogramming to naïve pluripotency for unraveling critical gene regulatory mechanisms acting during early mammalian development and highlighting prospects for new platforms for human disease and developmental modelling.

The availability of well-characterised monoclonal antibodies (mAbs) detecting cell-surface epitopes on human pluripotent stem cells (hPSCs) provides useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. We recently described generation of seven new mAbs that detect cell surface proteins present on primed and naive human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. In addition, we report that subsets of the seven new mAbs are also immunoreactive to specific human somatic cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

The availability of well-characterised monoclonal antibodies (mAbs) detecting cell-surface epitopes on human pluripotent stem cells (hPSCs) provides useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. We recently described generation of seven new mAbs that detect cell surface proteins present on primed and naive human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. In addition, we report that subsets of the seven new mAbs are also immunoreactive to specific human somatic cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

In the last years, progress has been made regarding the differentiation of human pluripotent stem cells into neural stem cells and astrocytes, growing into neurospheres and cerebral organoids. These models offer an exciting new range of opportunities to investigate developmental changes associated with zika virus (ZIKV) infection in human neural cells. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. Combining transcriptomics and proteomics, we showed that ZIKV alters the molecular fingerprint of neural stem cells by activating responses to viral replication, DNA damage targets, cell cycle arrest, apoptosis, as well as the downregulation of neurogenic programs. Our recent results will be discussed during the presentation.

Ductal cells share several markers with pancreatic embryonic progenitors, suggesting that ductal cells could be the closest lineage to an adult progenitor in the pancreas, thus their stem-like capacities in vitro have been under intense investigation. A commonly used assay for identifying stem or progenitor cells involves the isolation and culture of candidate cells under specific culture conditions to test their ability to generate colonies/spheres or organoids that can differentiate into multiple cell lineages either in vitro or following transplantation in vivo (Deleyrolle and Reynolds, 2009). Using such strategies, several cell types from the exocrine pancreatic compartment have been shown to acquire stem/progenitor-like properties in vitro (Rovira et al., 2010, Seaberg et al., 2004, Huch et al., 2013, Baeyens et al., 2005).

These research efforts have demonstrated that ductal/centroacinar cells, from human or mouse origin, can form spheres in suspension culture as well as organoids that under specific growth factor conditions or after kidney capsule engraftment can differentiate into β cells (Rovira et al., 2010, Huch et al., 2013, Boj et al., 2015, Seaberg et al., 2004, Jin et al., 2013, Lee et al., 2013). These organoid cultures represent a useful tool to study pancreas biology in an untransformed system in vitro, from human and mouse pancreatic ducts. This culture system together with new tools for genome editing holds promise for the study of pancreatic diseases and their progression (Huch et al., 2013, Huch and Koo, 2015, Hindley et al., 2016, Broutier et al., 2016).

Moreover, knowledge gained from studying the acquisition of stem-like/progenitor properties by some terminally differentiated pancreatic cell types in vitro can provide invaluable insight into harnessing these cells for regenerative therapy. Such studies can lead to defining the signaling pathways or growth factors whose activation in vivo is required to generate the proper niche that allow such population to act as dedicated or facultative progenitors.

Diabetes is a debilitating chronic disease that is spirally out of control. Fundamentally, the progressive failure of pancreatic beta cells results in decreased insulin secretion, ultimately giving rise to hyperglycaemia and overt diabetes. Given that there is a lack of access to pancreatic islets from diabetic patients with defined gene mutations or variants, the use of diabetic-patient-specific human induced pluripotent stem cells (hiPSCs) and their differentiation into pancreatic beta-like cells will provide an inexhaustible source of material for 1) in vitro disease modelling to study diabetes-related mechanisms, 2) developing small molecules that can enhance human beta cell replication and even 3) transplantation therapy.

Here, I will highlight our efforts in recruiting various types of diabetic patients, obtaining skin biopsies or peripheral blood mononuclear cells (PBMCs), deriving hiPSCs from these somatic cells and differentiating them into pancreatic cells. I will also provide an example of subjecting these diabetic-hiPSCs through a pancreatic differentiation protocol for in vitro disease modelling of diabetes. Overall, it will be evident that disease modelling of human diabetes via the use of diabetic-hiPSCs will provide novel insights into the development of diabetes and its complications.

Pluripotent stem cell (PSC) culture at commercial scale requires a shift from the current manual processing methods and costly materials to deliver affordable differentiated cell products in a repeatable and robust manner. The development of automated processing and analytical platforms for manufacturing allogeneic cell therapies derived from pluripotent stem cells is one of the aims of the Industrialisation group of the Cell and Gene Therapy Catapult (CGTC). This is centred around developing starting materials compliant with industry-standards, scalable 2D and 3D expansion systems as well as flexible downstream processing solutions. Here, we describe the current progress of CGTC’s Cell Plasticity platform programme with respect to developing automated processes for controlled expansion of pluripotent stem cells in 2D and 3D systems and strategies for culture intensification and integrated downstream processing solutions.

Acute kidney injury (AKI) is a serious public health problem worldwide, with ischemia and reperfusion (IR) being the main lesion-aggravating factors contributing to the evolution to a chronic kidney disease (CKD). The major intervention approaches available, hemodialysis and renal transplantation, are considered palliative options for the treatment of the disease. Face to this restricted scenario, the use of embryonic stem cells is suggested as an alternative, offering the possibility of replacement of affected cells and/or the release of paracrine factors crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways which are important for tissue regeneration, such as cell survival, proliferation, differentiation, and migration. Thus, the main objective of this work is to explore the protective potential of S1P and the activation pathway of its receptors (S1Pr1-5) in human embryonic stem cells (H9) and human kidney proximal tubule cells (HK-2) submitted to models of IR lesion, via depletion of ATP with Antimycin A (AA). S1P treatment decreased cell death after AA in both cell lines, and also decreased caspase-3 labeling in H9 cells. Nevertheless, S1P increased fluorescence intensity of Ki67 proliferation marker in HK-2 cells. HK-2 and H9 showed unaltered endogenous production of lipids, such as phosphatidylcholine (PC), phosphoethanolamine (PE) and lysophosphatidic acid (LPA), in the different conditions. However, the activity of the sphingosine kinase enzyme (SK), responsible for the production of S1P, was shown to be increased in H9 cells submitted to S1P + AA treatment compared to AA treatment. H9 showed expression of all five S1P receptors whereas HK-2 did not express S1Pr3. In addition, HK-2 showed increased fluorescence intensity for S1Pr1 after S1P + AA, and also for S1Pr2 after S1P. Inhibition of SK played an important role in both cell lines and a clear involvement of the JAK2 / STAT3 pathway was observed in H9 cells, using the AG-490 pathway inhibitor. In vivo treatment of male Wistar rats with a dose of 106 H9 cells via renal capsule injection showed improvement in renal parenchymal filling seen by SPECT after IR lesion. Mass spectrometric analysis of renal tissue was able to identify specific molecules in the IR renal cortex of treated animals compared to sham. Thus, we conclude that S1P, through the modulation of its receptors, seems to play an effective role in cellular protection in IR models of both H9 and HK-2 cells

Mitochondrial defects represent a common pathogenetic mechanism associated with neurodegeneration. At the same time, mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Addressing the mechanisms underlying mitochondrial impairment in patient-derived neural cells may therefore lead to the identification of therapeutic strategies counteracting neurodegeneration.

A critical element for cell-based drug discovery is the use of cells that are homogeneous and obtainable in a reproducible manner. We recently showed that NPCs derived from human induced pluripotent stem cells (iPSCs) represent an effective model system for mtDNA disease drug discovery (Lorenz et al, Cell Stem Cell 2017). Here, we present how we derive NPCs and post-mitotic neurons in a robust manner in order to use these cells for therapeutic screenings. Finally, we describe how to analyze the mitochondrial and metabolic properties of the patient-derived cells and how to set up high-content analysis (HCA)-based compound screening strategies.

For optimal use of human induced pluripotent stem cells (hiPSCs) it is essential to identify lines that are fully reprogrammed and of high quality with proven pluripotency in terms of differentiation. The ability to form teratomas in vivo is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal-dependent, laborious and only qualitative, there is an ongoing debate whether it is an acceptable tool. This has led to the development of an assay for quantitative analysis of teratomas (TeratoScore) as well as in vitro alternatives such as PluriTest and the hPSC Scorecard. We compared normal hPSCs, hiPSCs with reactivated reprogramming transgenes and nullipotent human embryonal carcinoma (hEC) cells in these assays. Cells were cultured on Vitronectin in TESR-E8 media (hPSCs) or in DMEM/10% FCS (hEC cells). The quality of undifferentiated cells was analysed by FACS for OCT3/4 as well as karyotyping. As assessed by immunohistochemistry and immunofluorescent staining the normal hPSCs gave rise to typical teratomas whereas the xenografts of the hEC cells and the hiPSCs with the reactivated reprogramming transgenes were largely undifferentiated and displayed a malignant phenotype.

TeratoScore confirmed typical teratomas and tumors lacking differentiation but was unable to identify partially differentiated tumors. The hPSC Scorecard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analysed with PluriTest only hEC cells were identified as distinct. All other undifferentiated cells lines including hiPSCs with reactivated transgenes were undistinguishable and resembled normal hPSCs. We propose a combination of PluriTest and hPSC Scorecard for quantitation of pluripotency status and function of hPSCs used for in vitro purposes. By contrast, the Teratoma assay is the only method which is able to determine whether hPSCs could potentially develop a malignant phenotype, resulting in exclusion for any clinical application.

As reprogramming methodologies have become more reliable and efficient, corresponding improvements were needed in the characterization workflow as well; specifically in terms of the speed, efficiency, specificity and sensitivity of the methods used.  Molecular analysis platforms offer a quantitative, accurate, and fast alternative to current methods and have recently been utilized to qualify pluripotent stem cells (PSC). When characterizing stem cells, it is important to confirm that a cell line a)  has not acquired any genomic abnormalities during derivation or genetic engineering, b) has or (maintains) the  capacity to differentiate into all three germ layers, and c) maintains expression of pluripotency associated markers. In addition to the characteristics described above, it is highly desirable to be able to uniquely ID every parental and subsequent derivative cell line; especially in core facilities, stem cell banks, or cell therapy development.
Here we describe three characterization methods: PluriTest, KaryoStat, and ScoreCard,. PluriTest and KaryoStat leverage microarray based platforms that provide a comprehensive gene expression profile to determine the state of pluripotency and whole genome coverage to detect chromosomal abnormalities via copy number variations, respectively. ScoreCard utilizes a focused panel of TaqMan qPCR based assays to assess expression of specifically selected genes that confirm pluripotency and differentiation potential.  These three methods provide a complete solution to fully characterize PSC lines for pluripotency, differentiation potential, genomic integrity and sample ID. 

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering.  The delivery of Cas9 plasmid DNA or mRNA involves in transcription and/or translation.  On the other hand, the direct delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) approves to be more effective.  In this endeavor, we have developed robust methods to purify and delivery Cas9 RNPs into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection.  Recently, we enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.  Under optimal conditions, we achieved precise genome editing rates of up to 24% in induced pluripotent stem cells (iPSCs) and 40% in Cas9-expressing iPSCs for a single nucleotide substitution at multiple genomic loci.   One of the keys to high HDR efficiency was placement of the cleavage site in close proximity to the intended site of editing.  Secondarily, asymmetric PAM and non-PAM single stranded (ss) DNA donors were also found to enhance HDR efficiency.   Taken together, we provide simple and highly efficient approaches for modulation of the mammalian genome and for generation of knock-in and knock-out cell lines.

Finding a job is a daunting and stressful process. Although technology has made the job find and application process much easier – but it also brings new challenges with how to manage the new technology involved in job seeking. In many ways, LinkedIn, a powerful professional networking tool has revolutionized the way we find jobs. In this seminar, Ania Wronski, Field Application Scientist for cel biology and former president of the Tufts Postdoctoral Association will provide advice as to how to use LinkedIn for your job search and answer common questions and pitfalls.

Since 1993, Gibco™ B-27™ Supplement and Neurobasal™ Medium has been the trusted standard for a variety of neuronal culture applications, with citations in more than 11,000 publications. However, as the desire for more reliable and biologically relevant models has increased, so too has the necessity for a media system that can maintain and mature functional neurons over long periods of time in culture. To meet this need, we have developed the new Gibco™ B-27™ Plus Neuronal Culture System comprising a neuronal basal medium (Neurobasal™ Plus) and serum-free supplement (B-27™ Plus) that provides significant improvements for long term viability and functionality of primary and PSC-derived neurons in vitro.

This next-generation media system features an optimized formulation, upgraded manufacturing process, and more stringent quality control for raw materials and final product.

Performance of this system was evaluated by a number of criteria including neurite outgrowth, neuronal survival and functionality in primary rat, mouse and human PSC-derived neurons for long term cultures at both low and high cell density. Taken together the results demonstrate that our new B-27™ Plus Neuronal Culture System is a superior solution to the current trusted standards used for culturing primary neurons, and maturing and maintaining hPSC-derived neurons.

The development of the CRISPR/Cas9 gene-editing platform enables the rapid generation of new genetically modified stem cell models of human diseases, as well as providing new potential therapeutic treatments. One challenge has been the efficient and consistent delivery of a range of gene-editing tools into human Pluripotent Stem Cells (PSCs) across different culture systems. We developed the Lipofectamine™ Stem transfection reagent as a complementary option to electroporation to be able to deliver large DNA plasmids, RNAs and Ribonucleoprotein (RNP) complexes into human induced Pluripotent Stem Cells and Neural Stem Cells. The ability to co-deliver Cas9 RNP complexes along with ssDNA templates enables both the disruption of genes, as well as introduction of specific basepair changes by Homology Directed Repair. Data demonstrating improved cutting activity in iPSCs with the next generation of TrueCut™ Cas9 Protein v2 with synthetic TrueGuide™ sgRNAs will be presented. In addition to Neon™ electroporation, optimized transfection and subsequent single-cell clonal isolation in a StemFlex™ Medium workflow will be reviewed. Scientists interested in gene-editing to create stem cell models for basic research, drug discovery and translational medicine will discover how these new tools can accelerate their research programs.

For many decades, cell lines have been cultured in standard CO2 incubators at “normal” atmospheric oxygen concentrations of about 21%. But oxygen concentrations in the human or animal body are much lower, varying from as low as 1-2 % up to about 12%, depending on the tissue microenvironment. Oxygen concentration affects metabolism, proliferation, differentiation, disease progression and more. Controlling oxygen in vitro is increasingly important in biological function and disease modeling, pharmaceutical development and production, cell therapy, and of course in culturing stem cells. Creating hypoxia is critical to maintain pluripotency, control differentiation, limit oxidative damage and more. In this talk, we:

• Review history and advances in understanding oxygen concentration in vivo and how hypoxia-inducible factor-1 (HIF-1) modulates the cell’s response.
• Present examples demonstrating the effects of hypoxia on stem cells and primary cells, including better cell growth, less differentiation and less oxidative damage.
• Explain how “tri-gas” CO2 incubators provide a physiological oxygen environment.

Disease-specific human pluripotent stem cells (hPSCs) represent a new chance to unravel cellular and molecular mechanisms of neurological diseases. Along this line, we were among the first to demonstrate that PGD-derived hES cells and derivatives offer pertinent cellular models of neuromuscular diseases. Indeed, we showed that PGD-derived hES cells and derivatives which express the causal mutation leading to the neuromuscular disorder Myotonic Dystrophy type 1 (DM1), can be used to model DM1-specific pathological features in vitro. Furthermore these models when combined to a whole genome transcriptomic analysis can unravel new physiopathological mechanisms. In parallel to these mechanistic analyses, we demonstrated that DM1 hESCs-based models can be used for therapeutic screenings leading to the recent launching of  a clinical trial on DM1 patients.

Altogether, our DM1 studies participated to the demonstration that disease-specific hPSCs and their derivatives are effective models of neuromuscular diseases to decipher new pathological mechanisms and allow for drug discovery with potential clinical applications. Furthermore failures and successes in the DM1 program was instrumental in defining important roadblocks to overcome in order to successfully model neuromuscular pathologies using hPSCs : 1) the development of a collaborative environment to have access to other models of the pathology that will further validate the results obtained using hPSCs derived cells, 2) the careful analysis of appropriate controls (ideally isogenic ones) and 3) the development of differentiation protocols to obtain with high yield and robustness the cell population most relevant to the pathology of interest.
Our general objective is now to take advantage of this expertise to refine pathological modeling of neuromuscular disorders. Neuromuscular diseases are a heterologous group of pathologies characterized by muscular paralysis due to intrinsic muscular defects and/or impairment of neural circuits controlling movement often through to spinal motor neuron degeneration. In that sense Myotonic Dystrophy Type 1 and Spinal Muscular Atrophy, the two pathologies on which our group is focusing represent paradigmatic neuromuscular disorders. They are both caused by ubiquitously expressed mutations and characterized by defects in the motor neuron/muscle interconnection.

Research projects developed by my group, that is implanted in the institute ISTEM, is to better understand physiological mechanisms implicated in the development of neuromuscular diseases and subsequently to develop new therapeutic strategies. We have three main domains of research: i). improve the protocols of differentiation to convert efficiently and reliably cell types of interest, i.e. motoneurons and muscle cells; ii). To improve our knowledge on the communication between motoneurons and muscle cells by developing new cellular systems iii). apply these different tools to neuromuscular diseases with the aim to decipher physiopathological mechanisms and to identify new pharmacological strategies. My group is mainly interested in two neuromusuclar diseases: myotonic dystrophy type 1 and spinal muscular atrophy. I recently took the head of the INSERM Unit located in ISTEM, institute dedicated to the use of human pluripotent stem cells to develop new therapeutic strategies for monogenic diseases. Two main of applications are developed in ISTEM: cell therapy and drug screening. More recently, I have been elected president of the French Society for Stem Cell Research (FSSCR, www.fsscr.fr). The main objective of this society, launched in January 2017, is to underpin and federate short and long-term efforts to ensure the place of France as a leader in the domain of stem cell research.

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