SummaryCell banking, disease modeling, and cell therapy applications have placed increasing demands on hiPSC technology. Specifically, the high-throughput derivation of footprint-free hiPSCs and their expansion in systems that allow scaled production remains technically challenging. Here, we describe a platform for the rapid, parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions described exhibit properties associated with the ground state of pluripotency. The simplicity and robustness of the system allow for the high-throughput generation and rapid expansion of a uniform hiPSC product that is applicable to industrial and clinical-grade use.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti–PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an “off-the-shelf” source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a “cold” tumor “hot” by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.

Human induced pluripotent stem cells (iPSCs) represent a scalable source of potentially any cell type for disease modeling and therapeutic screening. We have a particular interest in modeling skeletal muscle from various genetic backgrounds; however, efficient and reproducible methods for the myogenic differentiation of iPSCs have not previously been demonstrated. Ectopic myogenic differentiation 1 (MyoD) expression has been shown to induce myogenesis in primary cell types, but the same effect has been unexpectedly challenging to reproduce in human iPSCs. In this study, we report that optimization of culture conditions enabled direct MyoD-mediated differentiation of iPSCs into myoblasts without the need for an intermediate step or cell sorting. MyoD induction mediated efficient cell fusion of mature myocytes yielding multinucleated myosin heavy chain-positive myotubes. We applied the same approach to dystrophic iPSCs, generating 16 iPSC lines from fibroblasts of four patients with Duchenne and Becker muscular dystrophies. As seen with iPSCs from healthy donors, within 36 hours from MyoD induction there was a clear commitment toward the myogenic identity by the majority of iPSCs in culture (50%-70%). The patient iPSC-derived myotubes successfully adopted the skeletal muscle program, as determined by global gene expression profiling, and were functionally responsive to treatment with hypertrophic proteins insulin-like growth factor 1 (IGF-1) and wingless-type MMTV integration site family, member 7A (Wnt7a), which are being investigated as potential treatments for muscular dystrophy in clinical and preclinical studies, respectively. Our results demonstrate that iPSCs have no intrinsic barriers preventing MyoD from inducing efficient and rapid myogenesis and thus providing a scalable source of normal and dystrophic myoblasts for use in disease modeling and drug discovery.

Human induced pluripotent stem cells (hiPSCs) hold enormous potential, however several obstacles impede their translation to industrial and clinical applications. Here we describe a platform to efficiently generate, characterize and maintain single cell and feeder-free (FF) cultured hiPSCs by means of a small molecule cocktail media additive. Using this strategy we have developed an effective multiplex sorting and high-throughput selection platform where individual clonal hiPSC lines are readily obtained from a pool of candidate clones, expanded and thoroughly characterized. By promoting survival and self-renewal, the selected hiPSC clones can be rapidly expanded over multiple FF, single-cell passages while maintaining their pluripotency and genomic stability as demonstrated by trilineage differentiation, karyotype and copy number variation analysis. This study provides a robust platform that increases efficiency, throughput, scale and quality of hiPSC generation and facilitates the industrial and clinical use of iPSC technology.

hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30+ cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.