4th Annual 24 Hours of Stem Cells™ virtual event

The emergence of technology for development of induced pluripotent stem cells (iPSCs) from somatic cells, such as skin and blood cells, has resulted in the ability of researchers to have limitless pool of iPSCs retaining the genetic make-up of the somatic cells from which they were derived.  In conjunction with tools for downstream gene editing, such as clustered regularly interspaced short palindromic-repeat (CRISPR)-Cas9 nuclease systems, iPSCs can be used generate (1) knock-outs to study the impact of genes on cellular processes, or (2) knock-ins to assess the impact of reversal of point mutations on diseased states, or furthermore for generation of reporter cell lines.  Briefly, CRISPR-Cas9 systems provide simple and efficient site-specific targeting that is accomplished by guiding Cas9 nuclease via a variable 20-base guide RNA sequence to the site for formation of a double stranded break. This break can then be repaired via non-homologous end joining (NHEJ) where small insertions or deletions are made in the gene of interest to knock-out its function or via homology-directed repair (HR) in which single nucleotide changes or knock-ins can be accomplished using a donor DNA template for repair.  Together iPSCs and CRISPR-Cas9 systems provide researchers with effective in vitro tools for assessing gene function, disease modeling, and regenerative therapy.  In this webinar, we discuss new technologies available from Thermo Fisher Scientific which support (1) efficient delivery of CRISPR Cas9 to PSCs, (2) improve cell survival following transfection while maintaining normal PSC properties, and (3) improve clonal cell survival following low density seeding of 1, 3, or 5 cells per well of 96-well plate.

Neurons derived from human pluripotent stem cells (hPSCs) and primary rodent neurons both are excellent resources for disease modeling and drug screening.  Human PSCs derived neural stem cells (NSCs) can be expanded in culture and further differentiated into mature neurons for various applications, however, these often contain mixed population of both differentiated neurons and undifferentiated NSCs. Due to the continuing proliferation of undifferentiated NSCs, very high cell densities and cell aggregation are usually observed during the differentiation of hPSC-derived NSCs which increase over time, posing challenges for long-term maintenance and downstream analysis.  Primary rodent neuronal cultures, while highly physiologically relevant, are often challenged by glial cell overgrowth, which may exacerbate assay and analysis issues.  Here we demonstrate the use of CultureOne(tm) - a new supplement which can reduce the proliferation of undifferentiated NSCs without negatively impacting the rate or extent of differentiation for hPSC-derived NSCs.  Further, we will demonstrate the ability to "tune" the glial cell population in primary rodent neuronal cultures.  The overall effect in both instances increases the relevant population of neurons in culture.  Experimental data presented in this webinar will  illustrate the functionality, morphology, and maturity of these neuronal cultures.

Developing therapies 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 cell models for these in vitro approaches.  We review current advances in genome engineering and how to leverage a portfolio of these tools to generate knock-out and knock-in models for use in target or compound identification.  Specifically, we demonstrate this approach with induced pluripotent stem cells (iPSCs) to build isogenic disease models, which can be further differentiated to various cell types of interest that 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.

The use of pluripotent stem cell-derived cell types for disease modeling, drug screening and regenerative medicine is an exciting area of activity in health research. Prior to the availability of pluripotent stem cells and differentiation methods, relevant and affected primary cells were difficult to obtain and frequently only accessible post-mortem. Additionally, the recent development of CRISPR/Cas9 based genome editing methods allow the creation of isogenic (matched) disease and control lines that differ only at a specific, disease-relevant locus, or to insert reporter constructs into PSC lines such that the reporter may be used to study disease biology in relevant cell types after differentiation. Recently, CCRM has created a standard protocol for efficient genome engineering in human pluripotent stem cells (hPSC). This talk will describe our protocol, and its application to the creation of a cell line that contains a voltage-sensitive fluorescent reporter that will be used to study cardiac function and disease.

Stem cells play critical roles in the development of organisms, as well as in the maintenance and repair of organs and tissues throughout adulthood.  Advancing our understanding of mechanisms that control stem cell behavior – in particular their two hallmark properties of self-renewal and differentiation into specialized cells – will enable these cells to be increasingly harnessed to repair tissues damaged by disease or injury.  Stem cells reside within specialized microenvironments or niches that present them with a spectrum of regulatory signals to control their behavior. In particular, the niche presents stem cells with a range of molecular cues, and it has also been become increasingly apparent that key biophysical features of the environment modulate the presentation of this biochemical information. For example, spatial and temporal variation in the presentation of cues is important information that can impact fate decisions and tissue structure. In addition, the tissue matrix can have variable bulk mechanical properties and surface topographical properties depending on how its assembled.
 
We have created several technology platforms to investigate these problems, and in particular to understand and control the differentiation of adult neural stem cells and human pluripotent stem cells into neurons. First, we are developing and harnessing optogenetics as a system to investigate how cellular signaling dynamics impact fate decisions. Second, we develop bioactive, synthetic material systems to investigate the effects of cell-matrix and cell-cell interactions on cellular function. Finally, we work towards translating the basic information that emerges from both of these efforts into safe, scaleable, fully defined, robust culture and implantation systems for stem cell based regenerative medicine efforts to treat human disease.

The mission of the California Institute for Regenerative Medicine (CIRM) is to accelerate stem cell treatments to patients with unmet medical needs.  With $3 billion in funding and 300 active programs, it is the world’s largest institution dedicated to helping people by developing cell therapies.  To accomplish its mission, CIRM has funded a breadth of activities spanning from basic research to translation to clinical trials.  In addition to individual grants, CIRM has created resources to help the stem cell community worldwide.  Some resources promote research and drug development using stem cells.  CIRM has established an iPSC Repository maintained by the Coriell Institute that is currently the largest publically accessible pluripotent stem cell bank in the world.  CIRM also has a genomics research initiative that applies cutting edge sequencing and bioinformatics approaches to stem cell research and therapeutic development.  Other resources established at CIRM promote the acceleration of stem cell discoveries into therapeutic development.  CIRM is in the process of setting up a system of translational and clinical centers to help researchers move their discoveries into clinical development.  This system includes 1) a Translating Center to support preclinical IND-enabling activities such as process development, safety testing, and cell manufacturing of stem cell therapy candidates, 2) an Accelerating Center that helps with clinical strategy, regulatory submission, and clinical trial management, and 3) an Alpha Clinics network that conducts clinical trials for stem cell related therapies.  In total these resources are designed to catalyze the flow of stem cell discoveries to the clinic, which can come from both inside and outside of California.  CIRM has supported 16 stem cell based clinical trials and the goal is to have 50.  Beyond an overview of CIRM’s programs, this presentation will touch on perspectives around developing stem cell therapies and regulatory hurdles.

Inherited retinal degenerative disorders such as retinitis pigmentosa are characterized by death of the light sensing photoreceptive neurons of the outer retina. Like the rest of the CNS, the retina has little capacity for endogenous regeneration, and as a result, photoreceptor cell death causes debilitating irreversible blindness. Gene augmentation has the potential to prevent photoreceptor cell death, while cell replacement could actually repopulate the retina with new functioning photoreceptor cells and restore vision. In this talk I will show how we are using patient-specific iPSCs to evaluate disease pathophysiology, test novel gene-based therapeutics and develop autologous photoreceptor cell replacement for the treatment of retinal degenerative blindness.
 

The generation of induced pluripotent stem cells (iPSCs) from adult easily accessible somatic tissues was introduced ten years ago. This technology has revolutionized our opportunities to study human disorders including complex genetics and develop novel therapeutic concepts for regenerative medicine. By differentiating iPSCs to different cell types and stages, we can gain insights into developmental and disease driving molecular networks. Yet, a discussed question of the iPSC model system is the extent to which they retain residual phenotypes from their precursor, which impacts on the study of transcription networks. Moreover, it is open to which degree somatic mutations impact on functional studies of human genetic disorders when patient-specific iPSCs are applied. Here, we will tackle this question in the course of studying healthy and diseased siblings with Tetralogy of Fallot, a frequent congenital heart defect of multigenic origin. Routine genetic screenings are mainly based on genetic material derived from either whole blood or cheek swap sampling. ps-iPSCs are typically derived from dermal fibroblast (skin biopsies) or whole blood derived immortalized lymphoplastoid cell lines (LCLs). We will discuss whole genome frequency and impact of somatic acquired mutations on gene expression and functional studies of complex genetic disorders using ps-iPSCs.
 

• Background: Applications and general workflow of PSCs
• Reprogramming with high efficiency and safety (Summary of current reprogramming methods, Introduction of Cytotune-iPS 2.0 Sendai Reprogramming Kit)
• Optimum PSCs culturing system: (Summary of current culturing system, Introduction of KnockOut Serum Replacement-MultiSpecies and Essential 8)
• Characterization and differentiation of PSCs
• Resources and tools

Culture systems for pluripotent stem cell (PSC) expansion enable generation of a nearly unlimited pool of cells for downstream differentiation, disease modeling, drug discovery, and therapeutic applications.  While a number of PSC feeder-free medium systems exist, there are many challenges encountered by stem cell scientists across the PSC workflow in today’s applications.  Here we sought to improve the robustness and versatility of traditional PSC culture medium systems identify and optimize critical medium components.  Through assessment of over 65 different formulations, an optimum medium composition was identified which provides compatibility across the PSC workflow from somatic cell reprogramming, PSC expansion, downstream differentiation, as well as providing support in stressful applications such as gene editing.  This system additionally provides versatility, allowing for every-other-day or weekend-free feed schedules and compatibility with a broad range of passaging reagents and matrices.  Together this system provides a robust next-generation stem cell medium system for today’s challenging PSC workflow needs.  For Research Use Only

My research program focuses on building the technology to generate diverse human cell-types for application in regenerative medicine and drug toxicology testing. While embryonic stem cells (ESCs) have the potential to generate thousands of distinct human cell-types, this vast array of lineage choices has made it difficult to efficiently differentiate ESCs towards any single desired fate. To more accurately guide ESC differentiation, we have delineated comprehensive roadmaps that describe how ESCs are diversified into a wide variety of endodermal and mesodermal cell-types. These roadmaps have enabled the successful generation of human liver cells and bone progenitors that can engraft in mouse models and respectively regenerate human liver tissue or bones in vivo; as such these ESC-derived tissue progenitors serve as potential sources of human cells for regenerative medicine or drug toxicology testing
 

Most discussions about pluripotent stem cells center around their promise for regenerative medicine.  The most remarkable quality of these cells is their ability to turn into all of the cell types in the body. This amazing power is driving all of the projects in our lab that range from treatment of human disease to rescue of endangered species. One of our projects is development of an autologous cell replacement therapy for Parkinson’s disease (PD).  We have used non-integrating Sendai virus to reprogram skin cells into induced pluripotent stem cells (iPSCs) from ten people afflicted with PD.  Since death of dopamine neurons in the brain leads to the motor symptoms of PD, we have developed methods for generating dopamine neurons from iPSCs and plan to use these cells for autologous transplants, which should eliminate the need for immunosuppression. On the other end of the spectrum, we are using iPSCs in an effort to save endangered species. We are generating iPSCs from fibroblasts stored in the Frozen Zoo^® at the San Diego Wild Animal Park.  We are focusing on the Northern White Rhino, which will be extinct in our lifetimes.  Only 3 members of this species remain alive.  But there are fibroblasts from 12 individuals stored in the Frozen Zoo, and we have made iPSCs from two so far. We are generating iPSCs from the rest, and plan to differentiate them into gametes- this has been achieved for mice, but we don’t expect it to be easy for rhinos.  If we succeed, and can make fertilized embryos in the lab, there are 6 females from the closely related Southern White Rhino species already at the zoo who will serve as surrogate mothers.  The astounding power of pluripotency has opened doors to new ideas; we’re just getting started.