Tail-tip Fibroblasts Research Articles

Determining epigenetic memory in kidney proximal tubule cell derived induced pluripotent stem cells using a quadruple transgenic reprogrammable mouse

The majority of nucleated somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). The process of reprogramming involves epigenetic remodelling to turn on pluripotency-associated genes and turn off lineage-specific genes. Some evidence shows that iPSCs retain epigenetic marks of their cell of origin and this “epigenetic memory” influences their differentiation potential, with a preference towards their cell of origin. Here, we reprogrammed proximal tubule cells (PTC) and tail tip fibroblasts (TTF), from a reprogrammable mouse to iPSCs and differentiated the iPSCs to renal progenitors to understand if epigenetic memory plays a role in renal differentiation. This model allowed us to eliminate experimental variability due to donor genetic differences and transfection of the reprogramming factors such as copy number and integration site. In this study we demonstrated that early passage PTC iPSCs and TTF iPSCs expressed low levels of renal progenitor genes and high levels of pluripotency-associated genes, and the transcriptional levels of these genes were not significantly different between PTC iPSCs and TTF iPSCs. We used ChIP-seq of H3K4me3, H3K27me3, H3K36me3 and global DNA methylation profiles of PTC iPSCs and TTF iPSCs to demonstrate that global epigenetic marks were not different between the cells from the two different sets of tissue samples. There were also no epigenetic differences observed when kidney developmental genes and pluripotency-associated genes were closely examined. We did observe that during differentiation to renal progenitor cells the PTC iPSC-derived renal cells expressed higher levels of three renal progenitor genes compared to progenitors derived from TTF iPSCs but the underlying DNA methylation and histone methylation patterns did not suggest an epigenetic memory basis for this.

Pold4 is dispensable for mouse development, DNA replication and DNA repair

The DNA polymerase delta (Pol δ), a heterotetramer of four subunits (Pol δ4), plays a pivotal role in DNA replication, as well as in DNA damage repair. Pold4, as the smallest subunit of Pol δ, is degraded in response to DNA damage or when entering into S-phase. This leads to the conversion of Pol δ4 to the trimeric complex Pol δ3. However, the contribution of Pold4 has not been fully elucidated in mammals. Cdm1, the Pold4 ortholog in Schizosaccharomyces pombe, is dispensable for cell growth and DNA damage repair, and there are no Pold4 orthologs in Saccharomyces cerevisiae. We previously generated a knockout mouse model of Pold3 and revealed its essential role in genome stability. Unexpectedly, we here found that Pold4 knockout mice are viable and fertile. In addition, Pold4 knockout mice do not exhibit any pathologic changes in the lung and spleen, tissues with the most abundant expression of Pold4. Moreover, Pold4 knockout mouse tail tip fibroblasts (TTF) exhibited normal cell growth, cell cycle, DNA replication, DNA damage and DNA repair capacity. These results suggested that Pol δ3 but not Pol δ4 may be responsible for these processes in normal cells. Interestingly, 19-month-old wild-type (WT) mice had tumors in the liver, while Pold4 knockout mice did not, and Pold4 knockout mice showed increased longevity. In further, this provided evidence suggested that Pold4 could be a potential novel target for lung carcinoma because its depletion does not affect normal cells but does affect cancer cells.

Effects of Recloning on the Telomere Lengths of Mouse Terc+/- Nuclear Transfer-Derived Embryonic Stem Cells.

Haploinsufficiency of genes that participate in telomere elongation and maintenance processes, such as telomerase RNA component (Terc) and telomere reverse transcriptase (Tert), often leads to premature aging-related diseases such as dyskeratosis congenita and aplastic anemia. Previously, we reported that when mouse Terc+/- tail tip fibroblasts (TTFs) were used as donor cells for somatic cell nuclear transfer (SCNT, also known as cloning), the derivative embryonic stem cells (ntESCs) had elongated telomeres. In the present work, we are interested to know if an additional round of SCNT, or recloning, could lead to further elongation of telomeres. Terc+/- TTFs were used to derive the first-generation (G1) ntESCs, followed by a second round of SCNT using G1-Terc+/- ntESCs as donor cells to derive G2-Terc+/- ntESCs. Multiple lines of G1- and G2-Terc+/- ntESCs were efficiently established, and all expressed major pluripotent markers and supported efficient chondrocyte differentiation in vitro. Compared with donor TTFs, telomere lengths of G1 ntESCs were elongated to the level comparable with that in wild-type ntESCs. Interestingly, recloning did not further elongate the telomere lengths of Terc+/- ntESCs. Together, our work demonstrates that while a single round of SCNT is a viable means to reprogram Terc haploinsufficient cells to the ESC state, and to elongate these cells' telomere lengths, a second round of SCNT does not necessarily further elongate the telomeres.

Changes in tumor suppressors and inflammatory responses during hydrogen peroxide-induced senescence in rat fibroblasts

Cell proliferation and senescence are processes induced by oxidative stress. In this study, we aimed to establish a cellular model of rapid proliferation and senescence of rat tail-tip fibroblasts by hydrogen peroxide (H2O2), a well-known oxidant. On this basis, changes in oxidative stress, inflammatory response and cell cycle of fibroblasts were studied. After H2O2 treatment, cell counting and flow cytometry results showed that 50 μM of H2O2 for 12 h and 100 μM for 8 h effectively promoted fibroblast proliferation, while 500 μM rapidly led to cell cycle arrest. In addition, stimulation with H2O2 at a concentration of 50 μM also promoted the inflammatory effects of the cells. At a concentration of 100 μM H2O2, the cellular antioxidant system began to collapse at 8 h and began to affect cellular activity. 500 μM of H2O2 at 4 h the levels of senescence-associated β-galactosidase, a marker of senescence and oxidative stress, were almost positive in fibroblasts. In addition, we found that the risk of fibroblasts carcinogenesis increased with increased H2O2 stimulation. The results of this study indicate that H2O2 can cause rapid proliferation and senescence of fibroblasts and that its mechanism of action may be mainly through influencing cellular antioxidant systems, cellular inflammatory responses and cell cycle.

Oncolytic reovirus-mediated killing of mouse cancer-associated fibroblasts

Oncolytic viruses, which mediate tumor cell-specific infection, resulting in efficient tumor cell killing, have attracted much attention as a novel class of anti-cancer biopharmaceutical agents. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment that strongly supports the growth, survival, and metastasis of tumor cells, suggesting that CAFs would have influence to the antitumor effects of oncolytic viruses; however, it remains to be fully evaluated whether oncolytic viruses affect the viabilities and properties of CAFs following treatment. Oncolytic reovirus, which is a non-enveloped virus that contains 10-segmented double-stranded RNA genome, shows efficient tumor cell lysis without apparent cytotoxicity to normal cells and has been tested worldwide in clinical trials against various types of tumors. In this study, we demonstrated that reovirus exhibited cytotoxicity against mouse primary CAFs isolated from subcutaneous tumors, but not against tail-tip fibroblasts. Infection with reovirus resulted in activation of caspase 3 and up-regulation of apoptosis-related gene expression, indicating that reovirus induced apoptosis of mouse primary CAFs. Intratumoral administration of reovirus induced apoptosis of mouse CAFs in the tumor. Taken together, these results indicate that reovirus has the potential to mediate antitumor effects by killing not only cancer cells but also CAFs.

Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.

Over the last decade, great achievements have been made in the field of direct epigenetic reprogramming, which converts one type of adult somatic cells into another type of differentiated cells, such as direct reprogramming of fibroblasts into cardiomyocytes, without passage through an undifferentiated pluripotent stage. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate cardiac fibrotic remodeling in a diseased heart. Furthermore, in vitro reprogramming of fibroblasts into cardiomyocyte-like cells provides new avenues to conduct basic mechanistic studies, to test drugs, and to model cardiac diseases in a dish. Here, we describe a detailed step-by-step protocol for in vitro production of induced cardiomyocyte-like cells (iCMs) from fibroblasts. The related procedures include high-quality fibroblast isolation of different origins (neonatal cardiac, tail-tip, and adult cardiac fibroblasts), retroviral preparation of reprogramming factors, and iCM generation by fibroblast reprogramming via retroviral transduction of Gata4, Mef2c, and Tbx5. A detailed written protocol will help many other laboratories, inexperienced in this area, to use and further improve this technology in their studies of cardiac regenerative medicine.

Abstract 210: Reprogramming Mouse Fibroblasts Into Cardiac Progenitor Cells Using Crispr Technique Targeting Endogenic Genes

Background and Objective: CRISPR tools that allow for precise manipulation of individual loci have not been used in generation of i nduced c ardiac p rogenitor c ells ( iCPC s). This study was designed to determine the feasibility and effectiveness of reprogramming fibroblasts into iCPC using CRISPR activation (CRISPRa) system. Methods: Tail-tip fibroblasts (TTFs) were isolated from Nkx2-5 cardiac enhancer GFP reporter mice. A gRNA pool targeting 17 progenitor genes was synthesized and transduced with dCas9-VP64 lentivirus into TTFs ( Fig.1A ). The phenotype of iCPCs was then characterized by immunostaining and FACS of progenitor markers. Finally, the cardiac-lineage differentiation potential of iCPCs was determined by immunostaining and electrophysiological assay under defined induction mediums. Results: iCPCs with GFP expression were formed in TTFs after transduction of CRISPRa targeting Isl1, Gata4, Baf60c, Tbx5 and Nkx2-5 (Fig.1B), while GFP was not activated by control virus. Cardiac progenitor markers were activated in iCPCs as shown by immunostaining (Fig.1C). The generation efficiency of Flk1-postive iCPCs induced by CRISPRa was ~60% as showed by FACS. iCPCs can be differentiated into cardiomyocytes as identified by immunostaining of cardiac-specific markers (Fig.1D). The iCPC-derived cardiomyocytes displayed spontaneous beating and showed cardiac action potentials (Fig.1E). Conclusion: The CRISPRa system is an efficient and specific way to generate iCPCs, which could provide a novel source of cells for cardiac regenerative medicine. Conversion of fibroblasts into cardiac progenitors poses a novel therapeutic option for repair of myocardial infarction.

Generation of inner ear hair cells by direct lineage conversion of primary somatic cells.

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

Acceleration of somatic cell reprogramming into the induced pluripotent stem cell using a mycosporine-like amino acid, Porphyra 334

Porphyra 334 (P334), a mycosporine-like amino acid (MAA), is a secondary metabolite found in diverse marine and terrestrial organisms and has several beneficial effects on fibroblast proliferation, wound healing, and antioxidant activity. Here, we report that P334 accelerates the cell reprogramming process of mouse tail-tip fibroblasts (TTFs) and human dermal papilla (HDP) cells into induced pluripotent stem cells (iPSCs). We found that P334 significantly improved the cell reprogramming efficiency by activating the tri-methylation of histone 3 lysine 4 (H3K4me3), which controls mesenchymal to epithelial transition (MET) during the reprogramming process. Thus, we found that P334 directly regulates epigenetic changes, providing an efficient approach for natural compound-based cell reprogramming.

SSEA-3 and 4 are not essential for the induction or properties of mouse iPS cells.

Stage-specific embryonic antigens (SSEA-1, 3, and 4) are carbohydrate antigens that have been used as markers of embryonic stem (ES) cells. However, the roles of these antigens in the establishment and maintenance of stemness of ES and induced pluripotent stem (iPS) cells are still poorly understood. This study investigated the biological and functional significance of globo-series glycolipids such as SSEA-3 and 4 in mouse iPS cells induced from tail-tip fibroblasts (TTFs) of α1,4Gal-T-knockout mice (lacking SSEA-3 and 4). These iPS cells were induced by retroviral transduction of four factors (Oct3/4, Sox2, Klf4, and c-Myc) into TTFs, and colonies were picked up. Morphologically, the colonies resembled ES cells and were positive for alkaline phosphatase and ES cell markers. Furthermore, in vitro-differentiated induction experiments after embryoid body formation revealed that some colonies derived from α1, 4Gal-T knockout mice were able to differentiate into three germ layers. Three germ layers were also observed in teratomas from iPS cells derived from α1,4Gal-T-knockout mice. These results suggest that SSEA-3 and 4 are not essential, at least for the establishment and maintenance of stemness of mouse iPS cells.

Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac reprogramming

Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1β signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors.

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Abstract 17189: Reprogramming of Fibroblasts Into Cardiac Progenitor Cells Using Crispr Activation System

Background and Objective: CRISPR tools that allow for precise manipulation of individual loci have not been used in generation of i nduced c ardiac p rogenitor c ells ( iCPC s). This study was designed to determine the feasibility and effectiveness of reprogramming fibroblasts into iCPC using CRISPR activation (CRISPRa) system. Methods: Tail-tip fibroblasts (TTFs) were isolated from Nkx2-5 cardiac enhancer GFP reporter mice. A gRNA pool targeting 17 progenitor genes was synthesized and transduced with dCas9-VP64 lentivirus into TTFs ( Fig.1A ). The phenotype of iCPCs was then characterized by immunostaining and FACS of progenitor markers. Finally, the cardiac-lineage differentiation potential of iCPCs was determined by immunostaining and electrophysiological assay under defined induction mediums. Results: iCPCs with GFP expression were formed in TTFs after transduction of CRISPRa targeting Isl1 , Gata4 , Baf60c , Tbx5 and Nkx2-5 (Fig.1B), while GFP was not activated by control virus. Cardiac progenitor markers were activated in iCPCs as shown by immunostaining (Fig.1C). The generation efficiency of Flk1-postive iCPCs induced by CRISPRa was ~60% as showed by FACS. iCPCs can be differentiated into cardiomyocytes as identified by immunostaining of cardiac-specific markers (Fig.1D). The iCPC-derived cardiomyocytes displayed spontaneous beating and showed cardiac action potentials (Fig.1E). Conclusion: The CRISPRa system is an efficient and specific way to generate iCPCs, which could provide a novel source of cells for cardiac regenerative medicine.

Generation of four Isl1 reporter iPSC lines from cardiac and tail-tip fibroblasts derived from Ai6IslCre mouse

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCi-Ai6IslCre74 and ATCi-Ai6IslCre80.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming.

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. Stem Cells 2017;35:1197-1207.

Tissue-engineered 3-dimensional (3D) microenvironment enhances the direct reprogramming of fibroblasts into cardiomyocytes by microRNAs.

We have recently shown that a combination of microRNAs, miR combo, can directly reprogram cardiac fibroblasts into functional cardiomyocytes in vitro and in vivo. Reprogramming of cardiac fibroblasts by miR combo in vivo is associated with improved cardiac function following myocardial infarction. However, the efficiency of direct reprogramming in vitro is relatively modest and new strategies beyond the traditional two-dimensional (2D) culture should be identified to improve reprogramming process. Here, we report that a tissue-engineered three-dimensional (3D) hydrogel environment enhanced miR combo reprogramming of neonatal cardiac and tail-tip fibroblasts. This was associated with significantly increased MMPs expression in 3D vs. 2D cultured cells, while pharmacological inhibition of MMPs blocked the effect of the 3D culture on enhanced miR combo mediated reprogramming. We conclude that 3D tissue-engineered environment can enhance the direct reprogramming of fibroblasts to cardiomyocytes via a MMP-dependent mechanism.

Reconstitution in vitro of the entire cycle of the mouse female germ line.

The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.