Midlateral Approach Replantation of a Distal Phalanx Second Toe Amputation in a 4-Year-Old Child: A Case Report.

CASE: A 4-year-old girl patient presented with complete amputation of the second toe close to the distal interphalangeal joint. Replantation was performed using a novel midlateral approach. The procedure used the dorsal subcutaneous vein on the foot as a graft for the artery. Four months postoperatively, the toe healed without any complication. The patient reported pain-free physical exercise without limitations in daily activity. CONCLUSION: This report demonstrates that this approach has the potential to provide a safe and viable alternative for treating toe amputations and offers advantages such as simplified artery identification, straightforward anastomosis, and improved vein graft harvesting.

Success Rates of Finger Revascularization and Replantation.

Background: Revascularization surgery has been reported to have a higher success rate than replantation due to sufficient venous return. However, in complex cases, success depends on a wide range of indications. This study aimed to investigate success rates in cohorts that included severe cases. Methods: This single-center, noninterventional, retrospective cohort study included 292 patients (349 digits) who underwent revascularization or replantation at our institution between January 2000 and December 2022. Sex, age, smoking history, comorbidities, affected digit, amputation level, complete or incomplete amputation, type of fracture and mechanism, artery diameter, needle, vein anastomosis in the revascularization subgroup, vein grafting, warm ischemic time, and outcomes were investigated and compared between the revascularization and replantation subgroups of the distal and proximal amputation groups. Results: In the distal amputation group, the arterial diameter in the revascularization subgroup was larger than that in the replantation subgroup (P < 0.05). In the proximal amputation group, the revascularization subgroup had a lower frequency of multiple amputations than the replantation subgroup (P < 0.05). Vein grafts were more frequently used in both revascularization subgroups than in the replantation subgroups (P < 0.05). However, the other injury severity indices were similar, and the success rates were not significantly different between the subgroups. Conclusions: The revascularization success rate was similar to that of replantation. Vein anastomosis or vein grafting to the veins should be advocated for revascularization in severe cases where skin bridges may not have sufficient venous return.

Results of an Adjustable Traction Method Using Surgical Gloves and K-Wires for the Treatment of Proximal Interphalangeal Joint Fracture Dislocation.

Purpose: This study aimed to evaluate an adjustable traction method using surgical gloves and Kirschner wires (K-wires) for proximal interphalangeal (PIP) fracture dislocations and examine the association between a reduction pin and range of motion (ROM), and between subluxation immediately after removal and ROM. Methods: Patients who underwent this surgical method for PIP joint dislocation fractures between 2003 and 2017 were included. We retrospectively investigated the postoperative results. We defined patients having surgery within 4 weeks after an injury as fresh cases and after 4 weeks as chronic cases. K-wires were inserted at the center of the proximal phalangeal head and the distal part of the middle phalanx to create a frame, and the finger of the surgical glove was used as a traction-force generator. We analyzed the association of ROM with each finger, age, presence of a reduction pin, and subluxation immediately after frame removal. Results: Overall, 37 fingers were included (27 acute and 10 chronic). The mean age of the participants was 40.0 years (range: 13-72 years). The mean follow-up period was 10.5 months (3-47 months). The final active ROM was -4.6°/94.6° (extension/flexion) for acute cases and -27.0°/73.5° for chronic ones. Active ROM was significantly better in patients with a reduction pin than in those without it. Subluxation immediately after frame removal was not associated with postoperative active ROM. Additionally, all PIP joints with subluxation that occurred immediately after frame removal achieved good joint congruity. Conclusions: The results of the adjustable traction method using surgical gloves and K-wires were satisfactory. Postoperative ROM did not decrease because of the additional reduction pin. Subluxation occurring immediately after frame removal did not affect the ROM, ultimately resulting in good joint congruity. Type of study/level of evidence: Therapeutic IV.

Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.

The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors.

BACKGROUND: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer. RESULTS: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes. CONCLUSION: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Analysis of distribution, collection, and confirmation of capacity dependency of small extracellular vesicles toward a therapy for liver cirrhosis.

BACKGROUND: The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. METHODS: sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. RESULTS: The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. CONCLUSION: In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.

Selection Bias in Avoiding Vein Graft in Replantation/Revascularization May Exist in Distal and Proximal Amputations, Respectively.

No difference in the success rate has been reported between the vein graft and non-vein graft groups in replantation/revascularization. However, this depends on a wide range of indications in difficult cases. This study aimed to investigate the selection bias in avoiding vein grafts. Methods: This is a single-center, noninterventional, retrospective cohort study comprising 229 patients (277 digits) who underwent replantation/revascularization between January 2000 and December 2020 at our institution. Sex, age, smoking history, comorbidities, affected side, level of amputation, complete or incomplete amputation, type of fracture and mechanism, diameter of the artery, needle, warm ischemic time, and results were investigated and compared between the subgroups with and without vein graft. Results were investigated between the subgroups with and without a vein graft in the distal and proximal groups. Results: In the distal group, the mean arterial diameter of the vein graft subgroup was larger than that of the non-vein graft subgroup [0.7 (0.1) mm and 0.6 (0.2) mm, respectively, P < 0.05]. In the proximal group, the vein graft subgroup had higher severity than the non-vein graft subgroup (comminuted fracture, 31.1% versus 13.4%; and avulsion or crush amputation, 57.8% versus 37.1%, respectively, P < 0.05). However, the success rate was not significantly different between the aforementioned subgroups. Conclusion: There was no significant difference between the vein graft and non-vein graft subgroups owing to the selection bias avoiding small arteries in the distal amputation and the absence of said bias in the proximal amputation.

Mid-lateral approach for revascularization of an amputated second toe: A case report.

The plantar or dorsal approach has been previously reported for the replantation or revascularization of a completely or incompletely amputated lesser toe. However, no reports exist describing an alternative approach for the replantation or revascularization of an amputated lesser toe, either complete or incomplete. We encountered a rare case of revascularization of an incompletely amputated second toe using a mid-lateral approach. The purpose of this case report was to describe the mid-lateral approach, which is novel in its nature for the replantation or revascularization of a completely or incompletely amputated lesser toe. A 43-year-old male was involved in a motor vehicle accident and had incomplete crush amputation of a second toe at the base of the nail, along with open dislocation of the distal interphalangeal (DIP) joint in the third toe. We performed artery-only revascularization of the second toe using a mid-lateral approach, with the patient in the supine position with his hip in flexion and external rotation. The postoperative course was uneventful, and the second toe was deemed viable. The Japanese Society for Surgery of the Foot (JSSF) standard rating system of the lesser toe was rated 90 and the Self-Administered Foot Evaluation Questionnaire (SAFE-Q) scored 100 in all the mentioned categories. The mid-lateral approach could be an option for the replantation or revascularization of an amputated lesser toe distal to the proximal interphalangeal (PIP) joint.

Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells.

Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.

Induced pluripotent stem cells from homozygous Runx2-deficient mice show poor response to vitamin D during osteoblastic differentiation.

Cleidocranial dysplasia (CCD) is a hereditary disorder associated with skeletal dysplasia and dental abnormalities. CCD arises from heterozygous loss of function mutations in the Runt-related transcription factor 2 (RUNX2) gene. Osteoporosis is often observed in CCD patients and conventional vitamin D supplementation is recommended. However, sufficient evidences have not been presented yet. This study investigated the role of RUNX2 in osteoblastic differentiation and sought to identify potential target genes for the treatment of osteoporosis associated with CCD, using induced pluripotent stem cell (iPSC) technology. We successfully established Runx2-/-, Runx2+/- and wild-type miPSCs from litter-matched mice and found poor Vdr expression in Runx2-/-cells. Significant down-regulation of osteoblastic differentiation in Runx2-/- miPSCs was observed. Gene expression array revealed unexpected results such as remarkable increase of Rankl expression and decrease of Vdr in Runx2-/- cells. Insufficient response to vitamin D in Runx2-/- cells was also observed. Our results suggest that RUNX2 functions as a regulator of Rankl and Vdr and thereby controls bone density. These findings also suggest that conventional vitamin D supplementation may not be as effective as previously expected, in the treatment of osteoporosis associated with CCD, and that inhibiting RANKL function might be worth considering as an alternative treatment strategy.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency.

Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domains enable next-generation reprogramming technology with engineered reprogramming factors.

Early reactivation of clustered genes on the inactive X chromosome during somatic cell reprogramming.

Reprogramming of murine female somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by X chromosome reactivation (XCR), by which the inactive X chromosome (Xi) in female somatic cells becomes reactivated. However, how Xi initiates reactivation during reprogramming remains poorly defined. Here, we used a Sendai virus-based reprogramming system to generate partially reprogrammed iPSCs that appear to be undergoing the initial phase of XCR. Allele-specific RNA-seq of these iPSCs revealed that XCR initiates at a subset of genes clustered near the centromere region. The initial phase of XCR occurs when the cells transit through mesenchymal-epithelial transition (MET) before complete shutoff of Xist expression. Moreover, regulatory regions of these genes display dynamic changes in lysine-demethylase 1a (KDM1A) occupancy. Our results identified clustered genes on the Xi that show reactivation in the initial phase of XCR during reprogramming and suggest a possible role for histone demethylation in this process.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Factors Associated With Surgical Intervention for Osteoarthritis of the Thumb Carpometacarpal Joint.

PURPOSE: This retrospective study aimed to analyze the initial clinical factors associated with surgical intervention for osteoarthritis of the thumb carpometacarpal (CMC) joint. METHODS: The study included patients who first visited our hand surgery clinic, were given the diagnosis of osteoarthritis of the thumb CMC joint between May 2012 and January 2015, and were observed for more than 3 years. Patients were classified into 2 groups according to whether they had undergone surgery during the follow-up period. The following variables were extracted and included in a bivariate analysis: sex, age, age at onset, disease duration, dominant hand, pain visual analog scale (VAS) scores at rest and during use, night pain, Eaton classification, use of an orthosis, number of injections, tender area, range of motion, grip strength, pinch strength, Kapandji abduction index, palmar abduction distance, grind test results, CMC joint shape on radiographs, dorsal subluxation ratio, volar tilt of the metacarpal, presence or absence of ossicles, and the surgeon who recommended the surgery. Variables with P values less than .05 in the bivariate analysis were included in a logistic regression model. RESULTS: The study included 80 thumbs of 48 patients. Pain scores at rest and during use, and the dorsal subluxation ratio were identified as factors significantly associated with surgical intervention in the bivariate analysis. The subsequent logistic regression analysis including these factors as explanatory variables also identified the VAS score at rest and dorsal subluxation ratio as significantly associated with surgical intervention. CONCLUSIONS: The VAS score at rest and the dorsal subluxation ratio at the first clinical visit were associated with the likelihood of future surgical intervention within 3 years in patients with osteoarthritis of the thumb CMC joint. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic IV.

[A case of Listeria meningitis with active ulcerative colitis and a review of literature in the Japanese cases].

A 53-year-old man who had been diagnosed with moderate ulcerative colitis and treated with mesalazine and glucocorticoid steroid was admitted due to fever of unknown origin and diarrhea. Intravenous feeding and treatment with cephem antibiotics were started, but the febrile reaction did not improve at all. Physical examination and various tests showed no specific symptoms, including headache or meningeal irritation. However, the blood culture test showed a positive result of Gram-positive bacilli. Thus, a lumbar puncture was performed and the patient was finally diagnosed with Listeria monocytogenes bacteremia and meningitis. Administration of intravenous meropenem and ampicillin led to the improvement of symptoms without any neurological sequelae. In addition, several cases with opportunistic infection of L. monocytogenes have been reported in recent years in cases of inflammatory bowel disease (IBD) during immunosuppressive therapy. Consequently, L. monocytogenes infection should be considered as one of differential diagnosis when patients present with IBD patient and are treated by biological or immunosuppressive agents with a fever of unknown origin.

Wrist Contracture Caused by Adhesion of the Extensor Carpi Radialis Tendon after Distal Radius Fracture: A Case Report.

Although distal radius fractures are common, wrist contracture caused by an extra-articular lesion after a distal radius fracture is seldom reported. We report a rare case of wrist contracture caused by adhesion of extensor carpi radialis brevis (ECRB) tendon after distal radius fracture. The patient was successfully treated with tenolysis of the ECRB tendon.

Utilization of a novel Sendai virus vector in ex vivo gene therapy for hemophilia A.

Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.

Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.