The mechanistic role of trisomy 8 in the development of myelodysplastic syndrome (MDS) remains poorly defined. Here, we generated a trisomy 8 mouse model by transferring a human chromosome 8 into murine embryonic stem cells and prospectively examined the effects on hematopoietic stem cells (HSC) by trisomy 8. The expression of inflammatory genes was enhanced, and hematopoietic programs mediated by transcription factors and polycomb repressive complex 2 (PRC2) were dysregulated in trisomy 8 HSC, which impaired their self-renewal and balanced differentiation. Trisomy 8 HSC altered the chromatin accessibility and conformations and activated Y chromosome genes, such as Uty/Kdm6c epigenetic modifier, which is known to demethylate histone H3K27me3 modification. The Uty gene facilitated the activation of PRC2-target and Runx1-target genes in leukemogenesis and drove the proliferation of human trisomy 8 leukemic cells. Since the RUNX1 gene is frequently mutated in patients with trisomy 8 MDS, its deletion attenuated the enhanced expression of inflammatory genes and mitigated the impaired self-renewal of trisomy 8 HSC in mice. Our findings reveal that trisomy 8 altered the transcriptional programs and chromatin conformations in HSC and drove a pre-malignant state through activating the expression of Uty, suggesting a route for the development of trisomy 8 MDS.

The epigenetic landscape proposed by Waddington has long provided a conceptual framework for cellular differentiation [1]. In this model, pluripotent cells positioned at the summit of a developmental landscape progressively descend toward terminally differentiated states. The discovery of induced pluripotent stem (iPS) cells demonstrated that differentiated somatic cells can be reprogrammed back to pluripotency, revising the concept of irreversibility [2]. Emerging evidence suggests that rejuvenation does not necessarily require a return to this summit. Rather, partial reprogramming may restore youthful cellular properties while preserving lineage identity [3–5]. Here, I propose a conceptual framework in which rejuvenation occurs along the slope of the Waddington landscape. In this view, cellular identity and cellular aging represent partially separable dimensions of cell state. In mesenchymal stromal cells (MSCs), chemical, mechanical, and transient gene-expression strategies converge on restoration of youthful function without complete dedifferentiation [7–12]. This perspective suggests that partial reprogramming may enable the generation of transcriptionally diverse MSC populations, providing a conceptual basis for precision regenerative medicine.

BackgroundMesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but their clinical utility is limited by replicative senescence. Strategies that reverse aging while maintaining MSC identity are urgently needed.MethodsWe developed a non-integrating, temperature-sensitive Sendai virus (SeV)-mediated rejuvenation protocol transiently expressing hTERT, BMI1, and SV40T in human MSCs. Following SeV removal, we evaluated proliferation, telomere length, karyotype stability, transcriptomic reset, producing heterogeneity, and differentiation potential.ResultsRejuvenated MSCs (rej-MSCs) demonstrated extended proliferation beyond 100 days, telomere elongation, and normal karyotypes after SeV clearance. Transcriptomic profiling showed a reset of senescence-associated programs while retaining mesenchymal identity. Functional analyses revealed clone-specific heterogeneity, including HGF-driven angiogenic activity. Multilineage differentiation capacity was preserved across rej-MSCs.ConclusionsThis transient, non-integrating rejuvenation strategy establishes an operational definition of rej-MSCs and provides a transcriptionally diverse and scalable platform for MSC manufacturing and precision therapy design.

BACKGROUND/AIM:This study aimed to investigate whether matrix metalloproteinase-1 (MMP-1) expression serves as a poor prognostic factor in patients with ovarian cancer. PATIENTS AND METHODS:A total of 74 patients who underwent surgery for ovarian cancer at Tottori University Hospital between 2017 and 2022 were retrospectively analyzed. Preoperative serum MMP-1 levels were measured using a sandwich ELISA, and tumor tissue expression of MMP-1 was evaluated using immunohistochemistry. Associations between MMP-1 levels and clinical outcomes, including overall survival (OS) and progression-free survival (PFS), were assessed using Kaplan-Meier and Cox regression analyses. RESULTS:Elevated serum MMP-1 concentration was identified as an independent poor prognostic factor for overall survival (OS). However, MMP-1 expression in tumor tissue was not an independent prognostic factor for either OS or progression-free survival (PFS). Preoperative serum MMP-1 levels showed a positive correlation with Cancer Antigen 125 (CA125), indicating that the combination of these markers may be useful for predicting prognosis. CONCLUSION:Serum MMP-1 concentration may serve as a poor prognostic biomarker in ovarian cancer. Its use alongside CA125 could enhance prognostic assessment.

We aimed to generate immortalized stromal cell lines from the ovarian and fallopian tube tissues of a single patient using Sendai virus (SeV) vectors and identify candidate stromal genes involved in ovarian carcinogenesis. Tissues were collected from a 48-year-old woman with endometrioid borderline tumors and endometriomas. Primary cultures were established from the right ovarian endometrioma, left ovarian surface, bilateral fallopian tube, and endometrial surface. Immortalization was achieved using SeV vectors encoding human telomerase reverse transcriptase (TERT), B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), and Simian virus 40 large T antigen (SV40T). Morphologically, the established cells exhibited spindle-shaped fibroblast-like features and expressed stromal markers (Vimentin-positive, Keratin-negative), confirming their stromal origin. Genetic and molecular changes associated with immortalization were evaluated via chromosomal analyses, transcriptome sequencing, and reverse transcription-polymerase chain reaction (RT-PCR). SeV-infected stromal cell lines retained their proliferative capacity for over 25 passages, whereas non-infected primary cells lost their epithelial characteristics and underwent senescence after five passages. Chromosomal abnormalities were more prevalent in stromal cells derived from the ovarian endometriomas, suggesting early genomic instability. Transcriptomic profiling and RT-PCR revealed upregulation of matrix metallopeptidase 1 (MMP1), pregnancy-associated plasma protein A (PAPPA), and C-X-C motif chemokine ligand 1 in cyst-derived stromal cells compared to those from the normal ovary and fallopian tube, implicating these genes in extracellular matrix remodeling and tumor–stroma crosstalk. We established immortalized ovarian and fallopian tube stromal cell lines using SeV-based vectors. The cyst-derived stromal cells exhibited early chromosomal instability and overexpression of MMP1 and PAPPA, supporting their potential role in ovarian carcinogenesis. These immortalized stromal cell lines provide a novel and stable platform for mechanistic studies and may contribute to biomarker discovery and therapeutic target development in ovarian cancer.

Ulcerative colitis (UC) is an incurable intestinal disease, with current treatments mainly focused on inflammation control and, in severe cases, surgical resection. Recent studies have highlighted the need for new therapies that promote tissue regeneration. R-spondin-1 (RSPO1) and interleukin-22 (IL-22) have shown anti-inflammatory and regenerative effects in UC models, but have short half-lives and poor targeting abilities. Another therapeutic tool, mesenchymal stem cells (MSCs), offer promising migratory and homing capabilities; however, the preparation of homogeneous therapeutic MSCs at sufficient levels in vitro is challenging. Therefore, we developed a novel line of MSCs (HAC-MSC) with significant therapeutic effects in dextran sodium sulfate (DSS)-induced colitis mice. Construction of HAC-MSC involved a two-part strategy: 1) establishment of a non-integrating human artificial chromosome (HAC) vector carrying therapeutic genes encoding IL22 and RSPO1; and 2) transfer of the HAC to previously characterized rejuvenated MSCs (rej-MSCs) prepared using Sendai virus technology for prolonged proliferation capacity in vitro. HAC-MSC stably and efficiently produced therapeutic factors in vitro and, following intraperitoneal administration to DSS-induced colitis mice, showed continuous expression of the therapeutic factors over 5 days. Additionally, HAC-MSC-treated mice showed alleviation of the disease activity index score, reduced depth of injury, and promotion of intestinal growth compared with MSC-treated mice. Furthermore, effective treatment with HAC-MSC required only a fraction (1 %–10 %) of the number of cells needed for conventional MSC therapy. These findings highlight the outstanding potential of rej-MSCs carrying therapeutic factor-loaded HACs as a cell therapy tool with prospective applications in the treatment of UC and other diseases.

Human mesenchymal stem cells (hMSCs) with extended lifespan and differentiation potential that can recapitulate in vivo characteristics could significantly contribute to basic research, drug development, and cell therapy. Specifically, they could ensure a stable supply of specific cellular resources, and possibly extracellular vesicles. Here, we established a technology for extending the lifespan while maintaining differentiation potential, termed "rejuvenation," of hMSCs (rej-hMSCs) using nonintegrative and conditionally removable temperature-sensitive Sendai virus (SeV) vectors. Various immortalizing factors (i.e., Bmi-1, hTERT, SV40T, and/or HPV E6/E7) were first introduced by the SeV vector into the cells. A combination of three SeVs with Bmi-1, hTERT, or SV40T conferred markedly improved cell proliferation and cloning ability while maintaining differentiation potential and a normal karyotype. An extended lifespan was also demonstrated in other cell types. The rejuvenation of long-passaged or aged hMSCs was also confirmed. SeV vectors were rapidly removed as a function of cell doubling by increasing the temperature from 35 degrees C to 37 degrees C or higher, while proliferative ability was maintained. Following FACS sorting, the complete removal of SeV vectors was confirmed by qPCR analyses. Therefore, our cell rejuvenation technology could contribute to research and clinical applications by enabling the supply of modified cells without damaging host chromosomes.

The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1-2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications.

We aimed to elucidate the mechanism underlying carcinogenesis by comparing normal and BRCA1/2-mutated ovarian epithelial cells established via Sendai virus-based immortalization. Ovarian epithelial cells (normal epithelium: Ovn; with germline BRCA1 mutation: OvBRCA1; with germline BRCA2 mutation: OvBRCA2) were infected with Sendai virus vectors carrying three immortalization genes (Bmi-1, hTERT, and SV40T). The immunoreactivity to anti-epithelial cellular adhesion molecule (EpCAM) antibodies in each cell line and cells after 25 passages was confirmed using flow cytometry. Chromosomes were identified and karyotyped to detect numerical and structural abnormalities. Total RNA extracted from the cells was subjected to human transcriptome sequencing. Highly expressed genes in each cell line were confirmed using real-time polymerase chain reaction. Immortalization techniques allowed 25 or more passages of Ovn, OvBRCA1, and OvBRCA2 cells. No anti-EpCAM antibody reactions were observed in primary cultures or after long-term passages of each cell line. Structural abnormalities in the chromosomes were observed in each cell line; however, the abnormal chromosomes were successfully separated from the normal structures via cloning. Only normal cells from each cell line were cloned. MMP1, CCL2, and PAPPA were more predominantly expressed in OvBRCA1 and OvBRCA2 cells than in Ovn cells. Immortalized ovarian cells derived from patients with germline BRCA1 or BRCA2 mutations showed substantially higher MMP1 expression than normal ovarian cells. However, the findings need to be validated in the future.

Microcell-mediated chromosome transfer (MMCT) is anticipated as a unique strategy to manipulate numbers of chromosomes, including the generation of hyperaneuploidy syndrome models with human induced pluripotent stem cells (hiPSCs). Mouse A9/Chinese hamster ovary (CHO) cell libraries of human monochromosomal hybrids as chromosome donor cells frequently exhibit chromosomal rearrangement in the components. The generation of a new A9/CHO library is time-consuming and laborious. Here, we developed an MMCT method using hiPSCs as chromosome donor and recipient cells, through micronucleation using paclitaxel and reversine. Membrane fusion during the MMCT was mediated through interactions between the ecotropic viral envelope transiently expressed in chromosome donor cells and mCAT-1 in chromosome recipient cells. This approach involved tagging Chr21 and ChrY by CRISPR-Cas9 and transferring human/mouse artificial chromosomes, Chr21, ChrX, and ChrY, wherein there are no previous reports demonstrating a full-length introduction. Thus, a strategy that combing CRISPR-Cas9-mediated chromosome tagging and MMCT from hiPSCs as chromosome donor cells to hiPSCs as recipient cells systematically produced isogenic disease model hiPSCs with hyperaneuploidy. This approach allows the study of rare diseases and promises to provide new insights into early developmental mechanisms by introducing a comprehensive set of influential chromosomes/regions into hiPSCs.

14 Supplementary Figures and 2 Supplementary Tables

Supplementary Table 1 from Genetic Instability Caused by Loss of MutS Homologue 3 in Human Colorectal Cancer

Objective: We aimed to establish reversibly immortalized cell lines from human uterine and ovary cells using the Sendai virus (SeV) vector. The immortalized cells derive from normal and benign ovarian epithelial cells and endometrial epithelial cells. Furthermore, we sought to elucidate the mechanisms of carcinogenesis using the immortalized cell lines. Methods: Cells were collected at the time of surgery after obtaining patient consent. The cells used in this study were as follows: ovarian epithelial cells (normal epithelium, Ov n; normal epithelium with germline BRCA1 or BRCA2 mutation, Ov BRCA1 2; ovarian endometrioma, Ov endo; mucinous cystadenoma; Ov m), normal fallopian tube (FT) cells, and endometrial epithelium (normal epithelium, Em n). These cells were infected with temperature-sensitive SeV vectors carrying three immortalization genes, Bmi-1, hTERT, and SV40T.The presence of infection was confirmed through Green Fluorescent Protein (GFP) and Orange Fluorescent Protein (OFP). Immunoreactivity to the anti-human EpCAM antibody (a marker derived from epithelial carcinoma) in each SeV-infected cell was confirmed through flow cytometry. QH and multicolor FISH staining were performed for karyotyping of metaphase chromosomes in each cell line to determine chromosome number and structural abnormalities. Human transcriptome sequencing analysis was performed with NovaSeq 6000 (Illumina) using total RNA from each cell line. Some genes that showed significant expression in each cell line were subjected to real-time PCR (RT-PCR). Results: We established the immortalized cell lines from human uterine and ovarian tissues. SeV-infected cells exhibited GFP and OFP fluorescence, while non-infected cells did not. SeV infection allowed all primary cell lines to grow for 25 or more passages, while non-infected SeV cells lacked the proliferative capacity and showed senescence-like morphology. SeV-infected cells senesced in a temperature-dependent manner. Ov n SeV- infected cells ion causedshowed a small increase in chromosome structural abnormalities. But, Ov BRCA1 and 2 SeV-infected cells showed larger than thatand . Llong-term passaged cells did not show immune response to anti-human EpCAM antibodies in normal cells. Eleven Three genes were predominantly expressed in Ov BRCA1 and Ov BRCA2 cells and were not expressed in Ov n cells. SomeTwo out of ththe three genesem were found to be predominantly expressed in Ov endo cells compared to the expression in the Ovn cell line. Furthermore, RT-PCR results also indicated substantially higher expression of two genes in Ov BRCA1/2 cells and in Ov endo cells compared to the expression in Ov n cells. Conclusion: We succeeded in the reversible immortalization of endometrial and ovarian epithelial cells by using SeV infection. We identified several candidate genes that may be involved in the oncogenic mechanism of ovarian cancer associated with endometriosis or germline BRCA1 and BRCA2. Citation Format: Masayo Okawa, Hiroaki Komatsu, Kohei Hikino, Yuki Iida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Jun Chikumi, Shinya Sato, Genki Hichiwa, Yasuhiro Kazuki, Kanako Kazuki, Fuminori Taniguchi, Mitsuo Oshimura, Tasuku Harada. Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1214.

We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24–80) is more antigenic than the EpRE region (81–265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody–drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.

Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.

Myelodysplastic syndrome (MDS) is a poor prognosis cancer that predominantly affects the elderly, arising from hematopoietic stem cell (HSC), resulting in bone marrow failure and predisposition to acute myelogenous leukemia (AML). It has long been known that numerical chromosome anomalies such as trisomy 8 are critical for the development of MDS. Trisomy 8 is known to be associated with relatively poor prognosis, and is a criterion for the diagnosis of MDS in patients. Systemic inflammation has been implicated in the selective advantage for clonal hematopoietic stem cells and the progression of MDS, such as trisomy 8 MDS associated with Behcet's disease. Next generation sequencing studies revealed that trisomy 8 MDS cells highly mutated RUNX1 transcriptional factor (TF) and ASXL1, an epigenetic modifier, both of which regulate HSC function and the differentiation, relative to MDS cells without trisomy 8, suggesting that those mutations may help trisomy 8 to drive the development of MDS. However, previous clinical studies failed to identify genes on the chromosome 8 commonly amplified among trisomy 8 MDS patients, which may account for the pathogenesis of MDS, the molecular mechanism of development of trisomy 8 MDS remains unclear. Thus, we hypothesized that trisomy 8 initiates the transformation by multiple malfunctions of epigenetic and transcriptional regulators in HSC other than a gene dosage effect due to the extra chromosome 8. In order to determine how trisomy 8 affects hematopoiesis, we established a new trisomy 8 model by introducing a human chromosome 8 to mimic trisomy 8 in mouse ES cells. We succeeded in generating trisomy 8 chimeric mice and confirmed the mice showing emergence of hematopoiesis in fetal liver but significantly decreased the chimerism of trisomy 8 cells than the control mice. Wecompetitively transplanted trisomy 8 fetal liver cells at E14.5 into lethally-irradiated wild-type primary recipient mice, and moreover serial transplanted hematopoietic stem and progenitor cells (HSPCs) which isolated from the primary recipient mice. We found that trisomy 8 reduced the self-renewal capacity of HSPC and impeded the differentiation, but it was not sufficient to develop myeloid malignancies in mice. To elucidate the molecular mechanism underlying the impaired hematopoiesis by trisomy 8, we performed RNA sequencing in HSCs isolated from the primary transplanted mice. Gene set enrichment analysis revealed that in comparison with the control HSCs, trisomy 8 HSCs had significantly positive enrichments in type I and type II interferon response genes, but also the canonical target genes of Runx1 and PU.1 TFs were positively enriched in trisomy 8 HSCs. In addition, ATAC-seq analysis revealed that trisomy 8 HSCs changed chromatin accessibility enriched with the binding motif of Runx TF, compared to control HSCs. Given the changes in chromatin accessibility in trisomy 8 HSC, we performed chromatin conformation capture sequencing (Hi-C) in HSPCs and found that trisomy 8 induced changes in chromatin structures such as topologically associating domain (TAD) and compartment A/B in the other chromosomes, which were partly associated with changes in transcription of genes. Since the Runx1 gene is critical for HSC integrity and differentiation, and loss-of-function mutations of RUNX1 were often found in human myeloid malignancies with trisomy 8, we attempted to determine whether Runx1 deletion cancel the impaired self-renewal capacity of trisomy 8 HSC. We found that Runx1 deletion partially canceled the impaired repopulating capacity of trisomy 8 cells in recipient mice, suggesting the selective advantage of loss of function mutation of the RUNX1 gene in trisomy 8 mosaic people and patients with trisomy 8 MDS. Furthermore, as Janus kinase (JAK) and TANK-binding kinase 1 (TBK1) are critical for interferon signalling and activation of their downstream target genes, we revealed that the inhibition of either JAK or TBK function ameliorated trisomy 8-induced chromatin remodelling in HSCs. Overall, trisomy 8 changes chromatin structure and transcription of genes on other chromosomes in HSC accompanied with the malfunction of Runx1 TF, at least in part, due to the activation of interferon signaling, and results in the impaired hematopoiesis. We demonstrated that trisomy 8 creates the basis for transformation of MDS via remodeling chromatin structures in HSC.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Supplementary Figures 1-6, Methods and Materials from CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.