https://gladstone.org/u/dsrivastava/Articles/Ieda-Cell-2010.pdf

Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

RNA-guided gene activation by CRISPR-Cas9-based transcription factors

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. Heart failure affects over 14 million people worldwide and is a leading cause of death in adults and in children. Because postnatal cardiomyocytes (CMs) have little or no regenerative capacity, therapies are limited at present. The introduction of exogenous stem-cell-derived CMs holds promise, but also challenges, including delivery, integration, rejection and cellular maturation 1–3 . Reprogramming adult fibroblasts into induced pluripotent stem cells (iPSCs) that are similar to embryonic stem cells addresses some issues 4–6 , but others, including efficient directed differentiation into CMs and effective delivery, remain. A new generation of reprogramming technology involves transdifferentiating one adult somatic cell type directly into another 7–11 . We reported direct reprogramming of fibroblasts into CM-like cells in vitro by expressing three transcription factors: Gata4, Mef2c and Tbx5 (GMT) 7 . As observed in reprogramming to iPSCs, the percentage of fibroblast cells fully reprogrammed to beating CMs in vitro was small, but far more were partially reprogrammed, much like preiPSCs that can become fully pluripotent with additional stimuli 12 . We posited that cardiac fibroblasts may reprogram more fully in vivo in their native environment, which might promote survival, maturation, and coupling with neighbouring cells. If so, the vast pool of cardiac fibroblasts in the heart could serve as an endogenous source of new CMs for regenerative therapy.

/pdf/in-vivo-reprogramming-of-murine-cardiac-fibroblasts-into-omdgxrkryo.pdf

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

Transcriptional Regulation and Its Misregulation in Disease

Three papers in this issue demonstrate the production of functional induced neuronal (iN) cells from human fibroblasts, a procedure that holds great promise for regenerative medicine. Pang et al. show that a combination of the three transcription factors Ascl1 (also known as Mash1), Brn2 (or Pou3f2) and Myt1l greatly enhances the neuronal differentiation of human embryonic stem cells. When combined with the basic helix–loop–helix transcription factor NeuroD1, these factors can also convert fetal and postnatal human fibroblasts into iN cells. Caiazzo et al. use a cocktail of three transcription factors to convert prenatal and adult mouse and human fibroblasts into functional dopaminergic neurons. The three are Mash1, Nurr1 (or Nr4a2) and Lmx1a. Conversion is direct with no reversion to a progenitor cell stage, and it occurs in cells from Parkinson's disease patients as well as from healthy donors. Yoo et al. use an alternative approach. They show that microRNAs can have an instructive role in neural fate determination. Expression of miR-9/9* and miR-124 in human fibroblasts induces their conversion into functional neurons, and the process is facilitated by the addition of some neurogenic transcription factors. Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types1,2,3,4,5,6,7,8,9,10,11,12. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells13. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix–loop–helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

/pdf/induction-of-human-neuronal-cells-by-defined-transcription-537effsu7t.pdf

Induction of human neuronal cells by defined transcription factors

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

/pdf/direct-conversion-of-fibroblasts-to-functional-neurons-by-1ggt64l490.pdf

Direct conversion of fibroblasts to functional neurons by defined factors

Summary The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We found that the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction.

Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics.

<h3>Background</h3> Following a success of autologous chimeric antigen receptor (CAR)-T cells in hematologic malignancies, allogeneic CAR-transduced cells have been developed with various immune cells including induced pluripotent stem cell (iPSC)-derived NK cells and T cells. We have developed a novel platform of first-in-class iPSC-derived CAR-invariant natural killer T (iNKT) cells. iNKT cells are a rare subset of innate lymphocytes that bridge innate and adaptive immune response. iNKT cells can recognize and kill tumor cells, and further enhance antitumor activities of host endogenous immune cells by; cross-talking with dendritic cells; cross-priming tumor-specific CD8<sup>+</sup>T cells; transactivating NK cells; and reprograming pro-tumor myeloid cells. These intrinsic functions of iNKT cells possibly lead to high persistence and durability of allogeneic CAR-T cell therapy especially for solid tumors in the immunosuppressive tumor microenvironment. Usage of iPSC derived from iNKT cells is an ideal strategy to realize clinical scale production of functional iNKT cells from such a rare cell population. A Phase 1 study of the iPSC-derived non-transduced iNKT cells is currently ongoing in patients with head and neck squamous cell carcinoma. To demonstrate that CAR-transduced iPSC-derived iNKT cells provide a novel platform for effective cancer immunotherapy, the killing activities of CD19-CAR or HER2-CAR-transduced iPSC-derived iNKT cells were investigated in this first set of study. <h3>sMethods</h3> iNKT-derived iPSCs were engineered to express CD19-CAR or HER2-CAR by targeting the adeno-associated virus integration site-1 locus using genome editing method. CAR-iNKT cells were obtained by differentiation from CAR-introduced iPSCs under feeder cells free culture condition. The expressions of CAR and iNKT cell surface markers were examined by flow cytometry analysis. <i>In vitro</i> cytotoxicity of CAR-iNKT cells against cancer cell lines was examined by xCELLigence® real-time cell analyzer. <h3>Results</h3> CD19-CAR or HER2-CAR molecules were continuously expressed on engineered CAR-iPSCs. Both CAR-iPSCs were successfully differentiated into iNKT cells in feeder cell-free culture system without losing CAR expression. CAR- iNKT cells showed similar phenotypic properties to iPSC-derived non-transduced iNKT cells. Cytotoxic assay revealed that CAR-iNKT cells possessed enhanced antigen-specific killing activities against target molecule expressing cancer cell lines, such as CD19 positive NALM6 (B cell leukemia) or HER2 positive U-2 OS (Osteosarcoma), compared with iPSC-derived non-transduced iNKT cells. <h3>Conclusions</h3> This study showed the first successful delivery of a CAR construct into iPS cells that differentiate precisely into iNKT cells with enhanced cytotoxicity. iPSC-derived CAR-iNKT cells is demonstrated to become a novel allogeneic cell therapy platform. <h3>Acknowledgements</h3> We would like thank Dr. Takahiro Aoki at RIKEN for helpful discussions. 

/pdf/290-a-novel-ipsc-derived-car-invariant-natural-killer-t-inkt-1v9qefuf.pdf

Yuko Kokubu

Papers

Direct conversion of fibroblasts to functional neurons by defined factors

Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics.

290 A novel iPSC-derived CAR-invariant natural killer T (iNKT) cell therapy platform for hematologic malignancies and solid tumors