Correction to: Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

The article Tet3 regulates cellular identity and DNA methylation in neural progenitor cells, written by Miguel R. Branco and C. Joana Marques, was originally published electronically on the publisher's internet portal.

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

miR-125b promotes MLL-AF9-driven murine acute myeloid leukemia involving a VEGFA-mediated non-cell-intrinsic mechanism.

The hematopoietic stem cell-enriched miR-125 family microRNAs (miRNAs) are critical regulators of hematopoiesis. Overexpression of miR-125a or miR-125b is frequent in human acute myeloid leukemia (AML), and the overexpression of these miRNAs in mice leads to expansion of hematopoietic stem cells accompanied by perturbed hematopoiesis with mostly myeloproliferative phenotypes. However, whether and how miR-125 family miRNAs cooperate with known AML oncogenes in vivo, and how the resultant leukemia is dependent on miR-125 overexpression, are not well understood. We modeled the frequent co-occurrence of miR-125b overexpression and MLL translocations by examining functional cooperation between miR-125b and MLL-AF9 By generating a knock-in mouse model in which miR-125b overexpression is controlled by doxycycline induction, we demonstrated that miR-125b significantly enhances MLL-AF9-driven AML in vivo, and the resultant leukemia is partially dependent on continued overexpression of miR-125b Surprisingly, miR-125b promotes AML cell expansion and suppresses apoptosis involving a non-cell-intrinsic mechanism. MiR-125b expression enhances VEGFA expression and production from leukemia cells, in part by suppressing TET2 Recombinant VEGFA recapitulates the leukemia-promoting effects of miR-125b, whereas knockdown of VEGFA or inhibition of VEGF receptor 2 abolishes the effects of miR-125b In addition, significant correlation between miR-125b and VEGFA expression is observed in human AMLs. Our data reveal cooperative and dependent relationships between miR-125b and the MLL oncogene in AML leukemogenesis, and demonstrate a miR-125b-TET2-VEGFA pathway in mediating non-cell-intrinsic leukemia-promoting effects by an oncogenic miRNA.

Generation of functional thyroid from embryonic stem cells.

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Altered cofactor regulation with disease-associated p97/VCP mutations.

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.

Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

NLRP7 affects trophoblast lineage differentiation, binds to overexpressed YY1 and alters CpG methylation.

Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs), suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7-a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis-causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7, we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1, an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development, functions not previously associated with members of the NLRP family.

PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells.

Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.

Bone marrow mesenchymal stem cells of the intrauterine growth-restricted rat offspring exhibit enhanced adipogenic phenotype.

OBJECTIVE: Although intrauterine nutritional stress is known to result in offspring obesity and the metabolic phenotype, the underlying cellular/molecular mechanisms remain incompletely understood. We tested the hypothesis that compared with the controls, the bone marrow-derived mesenchymal stem cells (BMSCs) of the intrauterine growth-restricted (IUGR) offspring exhibit a more adipogenic phenotype. METHODS: A well-established rat model of maternal food restriction (MFR), that is, 50% global caloric restriction during the later-half of pregnancy and ad libitum diet following birth that is known to result in an obese offspring with a metabolic phenotype was used. BMSCs at 3 weeks of age were isolated, and then molecularly and functionally profiled. RESULTS: BMSCs of the intrauterine nutritionally-restricted offspring demonstrated an increased proliferation and an enhanced adipogenic molecular profile at miRNA, mRNA and protein levels, with an overall up-regulated PPARγ (miR-30d, miR-103, PPARγ, C/EPBα, ADRP, LPL, SREBP1), but down-regulated Wnt (LRP5, LEF-1, ß-catenin, ZNF521 and RUNX2) signaling profile. Following adipogenic induction, compared with the control BMSCs, the already up-regulated adipogenic profile of the MFR BMSCs, showed a further increased adipogenic response. CONCLUSIONS: Markedly enhanced adipogenic molecular profile and increased cell proliferation of MFR BMSCs suggest a possible novel cellular/mechanistic link between the intrauterine nutritional stress and offspring metabolic phenotype. This provides new potential predictive and therapeutic targets against these conditions in the IUGR offspring.

DNA methylation of Runx1 regulatory regions correlates with transition from primitive to definitive hematopoietic potential in vitro and in vivo.

The transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is alternatively expressed from two promoters: the proximal (P2) prevails during primitive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although some transcription factor binding sites and cis-regulatory elements have been identified, a mechanistic explanation for the alternative promoter usage remains elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of hematopoietic development in vivo and during differentiation of murine embryonic stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the primitive to definitive transition at the P1 promoter. In vitro, hypomethylation, acquisition of active chromatin modifications, and increased transcriptional activity at P1 are promoted by direct interaction with HOXB4, a transcription factor that confers definitive repopulation status on primitive hematopoietic progenitors. These data demonstrate a novel role for DNA methylation in the alternative promoter usage at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro.

p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cells.

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state.

Embryonic stem cell-based mapping of developmental transcriptional programs.

The study of developmentally regulated transcription factors by chromatin immunoprecipitation and deep sequencing (ChIP-seq) faces two major obstacles: availability of ChIP-grade antibodies and access to sufficient number of cells. We describe versatile genome-wide analysis of transcription-factor binding sites by combining directed differentiation of embryonic stem cells and inducible expression of tagged proteins. We demonstrate its utility by mapping DNA-binding sites of transcription factors involved in motor neuron specification.

A critical role for endoglin in the emergence of blood during embryonic development.

Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFß superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng(+)Flk1(+) mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71(+)Ter119(+) erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Scl, Gata1, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng(+)Flk-1(+) progenitors coexpressed TGFß and BMP receptors and target genes. Furthermore, Eng(+)Flk-1(+) cells presented high levels of phospho-SMAD1/5, indicating active TGFß and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFß superfamily ligands BMP4, BMP2, or TGF-ß1. These data demonstrate that the E(+)F(+) fraction at E7.5 represents mesodermal cells competent to respond to TGFß1, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling.

Molecular functions of the LIM-homeobox transcription factor Lhx2 in hematopoietic progenitor cells derived from mouse embryonic stem cells.

We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells.

Expression levels of endoglin distinctively identify hematopoietic and endothelial progeny at different stages of yolk sac hematopoiesis.

Endoglin (Eng), an ancillary receptor of the transforming growth factor beta (TGFß) signaling pathway superfamily, has been well recognized for its important function in vascular development and angiogenesis since its discovery more than a decade ago. Recent studies show that this receptor is also critical for the emergence of blood during embryonic development, and that at E7.5, endoglin together with Flk-1 identifies early mesoderm progenitors that are endowed with hematopoietic and endothelial potential. These two lineages emerge in very close association during embryogenesis, and because they share the expression of the same surface markers, it has been difficult to distinguish the earliest hematopoietic from endothelial cells. Here, we evaluated the function of endoglin in hematopoiesis as development progresses past E7.5, and found that the hematopoietic and endothelial progenitors can be distinguished by the levels of endoglin in E9.5 yolk sacs. Whereas endothelial cells are Eng(bright), hematopoietic activity is primarily restricted to a subset of cells that display dim expression of endoglin (Eng(dim)). Molecular characterization of these subfractions showed that endoglin-mediated induction of hematopoiesis occurs in concert with BMP2/BMP4 signaling. This pathway is highly active in Eng(dim) cells but significantly downregulated in the Eng knockout. Taken together, our findings show an important function for endoglin in mediating BMP2/BMP4 signaling during yolk sac hematopoietic development and suggest that the levels of this receptor modulate TGFß versus bone morphogenetic protein (BMP) signaling.

Eomesodermin induces Mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of Activin.

The transcription factor Eomesodermin (Eomes) is involved in early embryonic patterning, but the range of cell fates that it controls as well as its mechanisms of action remain unclear. Here we show that transient expression of Eomes promotes cardiovascular fate during embryonic stem cell differentiation. Eomes also rapidly induces the expression of Mesp1, a key regulator of cardiovascular differentiation, and directly binds to regulatory sequences of Mesp1. Eomes effects are strikingly modulated by Activin signalling: high levels of Activin inhibit the promotion of cardiac mesoderm by Eomes, while they enhance Eomes-dependent endodermal specification. These results place Eomes upstream of the Mesp1-dependent programme of cardiogenesis, and at the intersection of mesodermal and endodermal specification, depending on the levels of Activin/Nodal signalling.

Anakinra in Sanfilippo syndrome: a phase 1/2 trial.

Sanfilippo syndrome is a fatal childhood neurodegenerative disorder involving neuroinflammation among multiple pathologies. We hypothesized that anakinra, a recombinant interleukin-1 receptor antagonist, could improve neurobehavioral and functional symptoms owing to its capacity to treat neuroinflammation. This phase 1/2 trial aimed to test the safety, tolerability and effects of anakinra on neurobehavioral, functional and quality-of-life outcomes in patients and their caregivers. The primary outcome was the percent of participants requiring a dose increase at week 8 or week 16. Secondary efficacy outcomes included a multi-domain responder index (MDRI). Twenty-three participants (6-26 years of age) were enrolled. Twenty continued treatment to week 8, and 15 (75%) required an increased dose at week 8 or week 16. There was an improvement in at least one domain in the MDRI in 18 of 21 (86%) at week 8 and in 15 of 16 (94%) at week 36. Seven participants withdrew (intolerability of daily injections and lost to follow-up) before week 36. Adverse events occurred in 22 of 23 (96%) participants, most commonly mild injection site reactions. No serious adverse events were related to anakinra. In conclusion, anakinra was safe and associated with improved neurobehavioral and functional outcomes, supporting continued investigation of anakinra in Sanfilippo syndrome and other mucopolysaccharidoses. ClinicalTrials.gov identifier: NCT04018755 .

Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain.

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1ß when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1ß could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1ß.

Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression.

During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.

Skeletal muscle cell protein dysregulation highlights the pathogenesis mechanism of myopathy-associated p97/VCP R155H mutations.

p97/VCP, a hexametric member of the AAA-ATPase superfamily, has been associated with a wide range of cellular protein pathways, such as proteasomal degradation, the unfolding of polyubiquitinated proteins, and autophagosome maturation. Autosomal dominant p97/VCP mutations cause a rare hereditary multisystem disorder called IBMPFD/ALS (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis), characterized by progressive weakness and subsequent atrophy of skeletal muscles, and impacting bones and brains, such as Parkinson's disease, Lewy body disease, Huntington's disease, and amyotrophic lateral ALS. Among all disease-causing mutations, Arginine 155 to Histidine (R155H/+) was reported to be the most common one, affecting over 50% of IBMPFD patients, resulting in disabling muscle weakness, which might eventually be life-threatening due to cardiac and respiratory muscle involvement. Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to study pathology's underlying molecular mechanism, perform drug screening, and investigate regeneration. Using R155H/+ patients' fibroblasts, we generated IPS cells and corrected the mutation (Histidine to Arginine, H155R) to generate isogenic control cells before differentiating them into myotubes. The further proteomic analysis allowed us to identify differentially expressed proteins associated with the R155H mutation. Our results showed that R155H/+ cells were associated with dysregulated expression of several proteins involved in skeletal muscle function, cytoskeleton organization, cell signaling, intracellular organelles organization and function, cell junction, and cell adhesion. Our findings provide molecular evidence of dysfunctional protein expression in R155H/+ myotubes and offer new therapeutic targets for treating IBMPFD/ALS.