Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory.

Anti-NKG2C/IL-15/anti-CD33 killer engager directs primary and iPSC-derived NKG2C+ NK cells to target myeloid leukemia.

Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of "adaptive" NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C+ NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C+ cells to target CD33+ cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33+ cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C+ iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.

Suppression of leukemia development caused by PTEN loss.

Multiple genetic or molecular alterations are known to be associated with cancer stem cell formation and cancer development. Targeting such alterations, therefore, may lead to cancer prevention. By crossing our previously established phosphatase and tensin homolog (Pten)-null acute T-lymphoblastic leukemia (T-ALL) model onto the recombination-activating gene 1(-/-) background, we show that the lack of variable, diversity and joining [V(D)J] recombination completely abolishes the Tcrα/δ-c-myc translocation and T-ALL development, regardless of ß-catenin activation. We identify mammalian target of rapamycin (mTOR) as a regulator of ß-selection. Rapamycin, an mTOR-specific inhibitor, alters nutrient sensing and blocks T-cell differentiation from CD4(-)CD8(-) to CD4(+)CD8(+), the stage where the Tcrα/δ-c-myc translocation occurs. Long-term rapamycin treatment of preleukemic Pten-null mice prevents Tcrα/δ-c-myc translocation and leukemia stem cell (LSC) formation, and it halts T-ALL development. However, rapamycin alone fails to inhibit mTOR signaling in the c-Kit(mid)CD3(+)Lin(-) population enriched for LSCs and eliminate these cells. Our results support the idea that preventing LSC formation and selectively targeting LSCs are promising approaches for antileukemia therapies.

A chemical approach facilitates CRISPRa-only human iPSC generation and minimizes the number of targeted loci required.

Aim: We explored the generation of human induced pluripotent stem cells (iPSCs) solely through the transcriptional activation of endogenous genes by CRISPR activation (CRISPRa). Methods: Minimal number of human-specific guide RNAs targeting a limited set of loci were used with a unique cocktail of small molecules (CRISPRa-SM). Results: iPSC clones were efficiently generated by CRISPRa-SM, expressed general and naive iPSC markers and clustered with high-quality iPSCs generated using conventional reprogramming methods. iPSCs showed genomic stability and robust pluripotent potential as assessed by in vitro and in vivo. Conclusion: CRISPRa-SM-generated human iPSCs by direct and multiplexed loci activation facilitating a unique and potentially safer cellular reprogramming process to aid potential applications in cellular therapy and regenerative medicine.

Combined chemical and CRISPRa-mediated approach leads to efficient generation of human iPSCs.

iPSC-derived natural killer cells expressing the FcγR fusion CD64/16A can be armed with antibodies for multitumor antigen targeting.

BACKGROUND: Antibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK cells is mediated by the IgG Fc receptor CD16A (FcγRIIIA). Preclinical and clinical studies indicate that increasing the binding affinity and avidity of CD16A for antibodies improves the therapeutic potential of ADCC. CD64 (FcγRI), expressed by myeloid cells but not NK cells, is the only high affinity IgG Fc receptor and is uniquely capable of stably binding to free monomeric IgG as a physiological function. We have reported on the generation of the FcγR fusion CD64/16A, consisting of the extracellular region of CD64 and the transmembrane and cytoplasmic regions from CD16A, retaining its signaling and cellular activity. Here, we generated induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as a potential adoptive NK cell therapy for increased ADCC potency. METHODS: iPSCs were engineered to express CD64/16A as well as an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein and differentiated into iNK cells. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells and examined. NK cells, ovarian tumor cell lines, and therapeutic monoclonal antibodies were used to assess ADCC in vitro, performed by a DELFIA EuTDA assay or in real-time by IncuCyte assays, and in vivo. For the latter, we developed a xenograft mouse model with high circulating levels of human IgG for more physiological relevance. RESULTS: We demonstrate that (1) iNK-CD64/16A cells after expansion or thaw from cryopreservation can be coupled to therapeutic antibodies, creating armed iNK cells; (2) antibody-armed iNK-CD64/16A cells can be redirected by added antibodies to target new tumor antigens, highlighting additional potential of these cells; (3) cytokine-autonomous activity by iNK-CD64/16A cells engineered to express IL-15RF; and that (4) antibody-armed iNK-CD64/16A cells thawed from cryopreservation are capable of sustained and robust ADCC in vitro and in vivo, as determined by using a modified tumor xenograft model with high levels of competing human IgG. CONCLUSIONS: iNK cells expressing CD64/16A provide an off-the-shelf multiantigen targeting platform to address tumor heterogeneity and mitigate antigen escape.

Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

BACKGROUND: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection. METHODS: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors. FINDINGS: We demonstrated that 3MICA/B CAR mitigates MICA/B shedding and inhibition via soluble MICA/B while simultaneously exhibiting antigen-specific anti-tumor reactivity across an expansive library of human cancer cell lines. Pre-clinical assessment of 3MICA/B CAR iNK cells demonstrated potent antigen-specific in vivo cytolytic activity against both solid and hematological xenograft models, which was further enhanced in combination with tumor-targeted therapeutic antibodies that activate the CD16 Fc receptor. CONCLUSIONS: Our work demonstrated 3MICA/B CAR iNK cells to be a promising multi-antigen-targeting cancer immunotherapy approach intended for solid tumors. FUNDING: Funded by Fate Therapeutics and NIH (R01CA238039).

Generation of T-cell-receptor-negative CD8αß-positive CAR T cells from T-cell-derived induced pluripotent stem cells.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αß+ CAR T cells that perform similarly to CD8αß+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αß+ T cells for a broad range of immunotherapies.

Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma.

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. RESULTS: ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. CONCLUSIONS: These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies.

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy.

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.

Continuous sorting of heterogeneous-sized embryoid bodies.

This paper presents a microfluidic device for sorting embryoid bodies (EBs) with large dynamic size ranges up to 300 microm. The proposed separation scheme utilizes appropriately spaced pillars within a microchannel to alter the fluid flow pathway, thus allowing particles of defined sizes to be diverted towards specific flow paths. We test the device functionality by separating polystyrene beads 90, 175 and 275 microm in diameter, demonstrating separation efficiencies approaching 100%. We then demonstrate for the first time on-chip separation of mouse EBs, which were separated into three size groups. The ability to extract specific size ranges of EBs will greatly facilitate their subsequent differentiation studies.

iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy.

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.

Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells.

Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic "universal" cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing.

GSK3 Inhibition Drives Maturation of NK Cells and Enhances Their Antitumor Activity.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664-75. ©2017 AACR.

Generation of skeletal muscle cells from pluripotent stem cells: advances and challenges.

Human pluripotent stem cells (hPSCs) possess unlimited proliferative potential while maintaining the ability to differentiate into any cell type including skeletal muscle cells (SMCs). hPSCs are amenable to genetic editing and can be derived from patient somatic cells, and thus represent a promising option for cell therapies for the treatment of degenerative diseases such as muscular dystrophies. There are unresolved challenges however associated with the derivation and scale-up of hPSCs and generation of differentiated cells in large quantity and high purity. Reported myogenic differentiation protocols are long, require cell sorting and/or rely on ectopic expression of myogenic master regulators. More recent advances have been made with the application of small molecules to enhance the myogenic differentiation efficiency and the identification of more selective markers for the enrichment of myogenic progenitors with enhanced regenerative potential. Here we review the field of myogenic differentiation and highlight areas requiring further research.

Myogenic differentiation of muscular dystrophy-specific induced pluripotent stem cells for use in drug discovery.

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.