CIRM tools and technologies: Breaking bottlenecks to the development of stem cell therapies

Abstract The California Institute for Regenerative Medicine (CIRM) has a mission to accelerate stem cell treatments to patients with unmet medical needs. This perspective describes successful examples of work funded by CIRM's New Cell Lines and Tools and Technologies Initiatives, which were developed to address bottlenecks to stem cell research and translation. The tools developed through these programs evolved from more discovery‐oriented technologies, such as disease models, differentiation processes, and assays, to more translation focused tools, including scalable good manufacturing processes, animal models, and tools for clinical cell delivery. These tools are available to the research community and many are facilitating translation of regenerative therapeutics today.

In this perspective, we will describe some examples of outcomes from these RFAs and elaborate on how tool-focused discovery programs can both directly and indirectly advance the field.

| IN VITRO DISEASE MODELS
The invention of induced pluripotent stem cells (iPSCs) coupled with directed differentiation protocols has enabled the study of previously inaccessible human cell types, including neurons, glia, and cardiomyocytes (CMs). By deriving these cell types in vitro from both patient and control iPSC, scientists are able to identify cellular and molecular differences that may account for or reflect disease mechanisms. This ability has opened the possibility of understanding the cellular and molecular basis of complex polygenic neurological diseases that have stymied medicine for decades. In addition, iPSC models can be used to develop cell-based assays to screen for disease-modifying drugs.
Schizophrenia strikes in the prime of life with devastating impacts upon patients and their families. Medications have evolved little in decades, address only a subset of symptoms and can lead to serious and permanent side effects. Patients must also endure a trial and error period to identify an effective and tolerable regimen. 15 Thus, treatment of schizophrenia represents a clear unmet medical need. Unfortunately, the etiology of schizophrenia remains poorly understood. 3 To address this, Dr Fred Gage, with NCL grant support, developed human induced pluripotent stem cell (hiPSC) lines from five unaffected controls and four patients with a history of schizophrenia with varying genetic backgrounds. After differentiating the cells to neurons in vitro, genes previously linked to schizophrenia in genome-wide association studies were confirmed to have altered gene expression in the patient samples, and new gene expression changes were also identified. Patient neurons showed fewer neurites and decreased connectivity, and these deficits were rescued by the antipsychotic drug loxapine. Taken together, these early observations suggest that hiPSC can be used to model a polygenic disorder and potentially to identify patient specific treatments. This line of inquiry is continuing to contribute to our understanding of disease. 16,17 3 | NOVEL ASSAYS

| In vitro pluripotency assay
The development of hiPSC and the explosion of new human pluripotent stem cell (hPSC) lines has given impetus to develop simpler and less expensive in vitro assays for pluripotency. The formation of teratomas after implantation in immune deficient mice has been the gold standard assay to assess pluripotency of hPSC lines 18

| Automated differentiation optimization
To take advantage of the potential of stem cells to give rise to specific desired cell fates, scientists typically develop differentiation protocols through an iterative trial and error process, experimenting with the timing and concentration of growth factor, cytokine, or small molecule

| Genetic modification of stem cells
The ability to edit genomes in pluripotent stem cells is a powerful way to study the function of specific genes during or after differentiation to spe-

| Xeno-free, scalable manufacturing of CMs
The ability to replace cardiac myocytes lost to a myocardial infarction has the potential to prevent progression to heart failure. At an approximate dose of 1e9 cardiac myocytes per patient, to be prac-

| Cell delivery to human brain
Adequate and accurate delivery of therapeutic intervention to targeted sites is critical for both safety and efficacy of cell and gene therapies. However, covering a large brain region, such as the putamen, with traditional methods requires multiple injections with a straight cannula. Each brain penetration increases the bleeding risks to the patient, and the resultant delivery pattern is constrained to lines of single, parallel needle tracks. To address these limitations, Dr Daniel Lim's award funded the development of the radial branched deployment delivery device (RBD). The device employs a collet with a side port through which a switchable cannula is placed. After entry through a single site, the surgeon can rotate the angle of the side port to deliver cells through the cannula at multiple angles at a given depth.
The depth of delivery can then be modified to achieve three-dimensional coverage of a targeted brain region in a tree like pattern. The RBD can also be used with existing magnetic resonance imaging compatible frames to allow real time targeting. In this way, the surgeon can achieve tailored coverage of a brain target. This patented technology achieved a 510K filing and is available to investigators for further testing. 36 6.3 | Xenobiotic-free matrices for cell delivery

| LESSONS AND OPPORTUNITIES
In this perspective, we have described examples ( marketed products and instruments, new technologies poised for clinical trials, and the launch of new for profit companies. These multidisciplinary investigators continue to work in the stem cell field and have collectively advanced regenerative medicine both by the tools they developed as well as the discoveries that were enabled by them.
The ultimate impact of this CIRM investment will continue to unfold in the coming years.