阿部 朋行

Luminescent technology based on the luciferin-luciferase reaction has been extensively employed across various disciplines as a quantitative imaging modality. Owing to its non-invasive imaging capacity, it has evolved as a valuable in vivo bioimaging tool, particularly in small animal models in fields such as gene and cell therapies. We have previously successfully generated rats with a systemic expression of the luciferase gene at the Rosa26 locus. In this study, we transplanted bone marrow from these rats into micro-mini pigs and used in vivo imaging to non-invasively analyze the dynamics of the transplanted cells. In addition, we established that the rat-to-pig transplantation system is a discordant system, similar to the pig-to-human transplantation system. Thus, rat-to-pig transplantation may provide a clinically appropriate large animal model for pig-to-human xenotransplantation.

Sheep are typically considered as industrial animals that provide wool and meals. However, they play a significant role in medical research in addition to their conventional use. Notably, sheep fetuses are resistant to surgical invasions and can endure numerous manipulations, such as needle puncture and cell transplantation, and surgical operations requiring exposure beyond the uterus. Based on these distinguishing characteristics, we established a chimeric sheep model capable of producing human/monkey pluripotent cell-derived blood cells via the fetal liver. Furthermore, sheep have become crucial as human fetal models, acting as platforms for developing and improving techniques for intrauterine surgery to address congenital disorders and clarifying the complex pharmacokinetic interactions between mothers and their fetuses. This study emphasizes the significant contributions of fetal sheep to advancing human disease understanding and treatment strategies, highlighting their unique characteristics that are not present in other animals.

This preliminary study investigated the efficacy and safety of bone marrow-derived mesenchymal stem cell transplantation in a piglet Legg-Calve-Perthes disease (LCPD) model. The LCPD model was induced in two Landrace piglets (6- and 7-week-old, weighing 12 and 17 kg, respectively) by ligaturing the femoral neck. In the first piglet, the natural LCPD course was observed. In the second piglet, 4 weeks after ligaturing the femoral neck, simple medium and medium containing 2.44 × 107 bone marrow-derived mesenchymal stem cells were transplanted into the right and left femoral heads after core decompression, respectively. Plain radiographs were obtained every 4 weeks, and the epiphyseal quotient was calculated by dividing the maximum epiphysis height by the maximum epiphysis diameter. The piglets were sacrificed at 14 weeks postoperatively. The femoral heads were extracted and evaluated grossly, pathologically, and by using computed tomography. The transplanted cell characteristics were evaluated using flow cytometry. Flattening of the epiphysis was observed in both femoral heads of the first piglet and only in the right hip of the second piglet. The epiphyseal quotients immediately and at 14 weeks postoperatively in the right femoral head of the second piglet were 0.40 and 0.14, respectively, while those of the left femoral head were 0.30 and 0.42, respectively. Hematoxylin and eosin staining did not reveal physeal bar or tumor cell formation. The transplanted cells were 99.2%, 65.9%, 18.2%, and 0.16% positive for CD44, CD105, CD29, and CD31, respectively. Core decompression combined with bone marrow-derived mesenchymal stem cell transplantation prevented epiphyseal collapse.

Abstract Background Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. Methods We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient’s F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. Results Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG–mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. Conclusion A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.

ヒツジといえば、多くの読者が羊毛あるいは羊肉のための産業動物を思い起こすであろう。ヒツジは医学研究を支える実験動物としても活躍している。ヒツジ胎仔には外科的侵襲に耐性があり、注射針穿刺による細胞移植や子宮外への露出を伴う外科処置などのさまざまな操作を行っても流産しにくい。このような性質をいかし、筆者らは胎仔肝臓内を介してヒトiPS細胞由来の血液を作るキメラヒツジの作出に成功した。また、ヒト胎児モデルとして活用し、先天性疾患に対する子宮内外科手術の手技開発・技術トレーニングや、母児間の薬物動態を解明する研究を行ってきた。本稿では、他の動物にはないヒツジ独特の性質に焦点を当てながら、人間の病気やその処置法の開発に貢献するヒツジを用いた先進医学研究を紹介する。(著者抄録)

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

多能性幹細胞由来の造血幹細胞の作成は,血液学における長年の課題であった.これに向けていくつもの技術が考案されてきたが,実用化に向けてまだ解決すべき課題が残されている.本稿では,試験管内培養技術や動物体内を利用した分化誘導技術に焦点を当てて,造血幹細胞作成技術の進歩や新たな知見について述べる.また,我々独自のヒツジ子宮内移植法による胎仔肝臓の利用,ヒト造血幹細胞の生着とその促進技術について紹介する.(著者抄録)

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP+ PSC-CMs exhibited more mature phenotypes than RFP- cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Human induced pluripotent stem cells (hiPSCs) are creating great expectations for regenerative medicine. However, safety strategies must be put in place to guard against teratoma formation after transplantation of hiPSC-derived cells into patients. Recent studies indicate that epigenetic regulators act at the initial step of tumorigenesis. Using gain-of-function and loss-of-function approaches, we show here that the expression and function of lysine-specific demethylase 1 (LSD1) are tightly regulated in hiPSCs, and their deregulation underlies the development of teratomas. Consistent with these results, we demonstrate that an LSD1 inhibitor, S2157, prevented teratoma formation from hiPSCs transplanted into immunodeficient mice. This novel action of LSD1 and the effects of its inhibition potentially allow for the development of new clinical applications and therapeutic strategies using hiPSCs.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20–100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=−0.97, P&lt 0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/− pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34(-)) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin(-) or 18Lin(-)) CD34(+/-) cells from human CB in mice and sheep. Both 13Lin(-) and 18Lin(-) CD34(+) cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin(-) nor 18Lin(-) CD34(-) cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin(-) and 18Lin(-) CD34(-) cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34(-) HSCs is significantly more limited than that of CD34(+) HSCs. As for 18Lin(-), CD34(-) HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34(-) HSCs could thus explain in part their reduced ability to home to the BM niche.

ヒト造血幹細胞(hematopoietic stem cells:HSC)は、免疫不全マウスを用いた造血再構築実験で評価されてきた。しかし、小型・短命のマウスでヒト造血を長期間評価するには限界があることから、大型動物を用いた評価法の確立が望まれる。これまでに我々は、免疫系が未発達なヒツジ胎仔にヒトHSCを移植することで、生後のヒツジ骨髄中においてヒト造血細胞(colony forming unit:CFU)の検出に成功した。今回、このヒツジ子宮内移植系を用いて、ヒト造血幹細胞の生着を長期にわたって評価した。本解析には14頭のヒツジを用いた。そのうち4頭では、HSC増幅遺伝子であるHOXB4をヒトHSCに導入し、移植した。別の4頭では、移植前処置剤であるブスルファンを妊娠ヒツジに投与した後で、ヒトHSCを移植した。移植後5ヵ月において、ヒトHSCにもヒツジにも処置を加えずに移植した6頭ではヒトCFUが検出されなかったが、何らかの処置を加えた残りの8頭中7頭では1-3%の割合でヒトCFUの生着を示した。さらに、HOXB4遺伝子を導入したヒトHSCを移植したヒツジでは、移植後40ヵ月にわたってヒトCFUの生着が維持された。以上より、ヒツジ子宮内移植系を用いることで、マウスの系のような植え継ぎ移植を行わずに、ヒトHSCの生着をヒツジ1個体内で長期的に評価できることが示された。(著者抄録)

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing JUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent JUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the JUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells. (C) 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Objective The homeobox B4 (HoxB4) gene promotes expansion of hematopoietic stem cells (HSCs) However, frequent development of leukemia in large animals duo to retrovirally transduced HoxB4 gene has been reported To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/Delta P), which enabled clearance of the vector and transgene shortly after transduction We tested the SeV/Delta P vector expressing the HoxB4 gene (SeV/Delta P/HoxB4) for the ex vivo expansion of human cord blood CD34(+) cells (HSCs) using a sheep in utero transplantation assay Materials and Methods Human HSCs were ex vivo expanded by transduction with SeV/Delta P/HoxB4 vector and transplanted into the abdominal cavity of fetal sheep The. engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony forming unit assays Results After transplantation, the HoxB4-transduced HSCs contributed to longer period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group (p < 0 05) The expansion of human HSCs with the SeV/Delta P/HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4 The SeV/Delta P/HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post transplantation Conclusions The SeV/Delta P vector would be suitable for transient expression of HoxB4 in human CD34(+) cells In addition, the SeV/Delta P vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors (C) 2011 ISFH Society for Hematology and Stem Cells Published by Elsevier Inc

In the present study, we investigated the suitability of two methods for the transplantation of cells into ovine fetuses. The first method was an ultrasound-guided cell injection via the uterine wall. The second involved hysterotomic cell injection with an incision in the uterine wall exposing the amnion. Monkey embryonic stem (ES) cell-derived hematopoietic cells were used as donor cells. After transplantation, the abortion rate associated with the hysterotomic injection method was significantly higher than that of the ultrasound-guided injection method (8/13 versus 4/24; P < 0.01). The fetuses were delivered to examine the engraftment of transplanted monkey hematopoietic cells. Monkey cells were detected in one of the five animals (20%) in the hysterotomic injection group, and 14 of 20 animals (70%, P < 0.05) in the ultrasound-guided injection group. Therefore, the ultrasound-guided method was effectively shown to be minimally invasive for in utero transplantation and can produce a higher rate of engraftment for transplanted cells.

Because embryonic stem (ES) cells are able to proliferate indefinitely and differentiate into any type of cell, they have the potential for providing an inexhaustible supply of transplantable cells or tissues. However, methods for the in vitro differentiation of human ES cells are still quite limited. One possible strategy would be to generate differentiated cells in vivo. In view of future clinical application, we investigated the possibility of using xenogeneic large animals for this purpose. We transplanted nonhuman primate cynomolgus ES cells into fetal sheep at 43-67 gestational days ( full term 147 days, n = 15). After birth, cynomolgus tissues, which were mature teratomas, had been engrafted in sheep when more than 1 X 10(6) ES cells were transplanted at < 50 gestational days. Despite the sustained engraftment, both cellular and humoral immune responses against the ES cells were detected, and additional transplantation was not successful in the animals. At 2 weeks post-transplantation, the ES cell progeny proliferated when transplanted at 48 ( < 50) gestational days, whereas they were cleared away when transplanted at 60 ( < 50) gestational days. These results support the rapid development of the xenogeneic immunological barrier in fetal sheep after 50 gestational days. Notably, a large number of Foxp3(+) regulatory T cells were present around the ES cell progeny, but macrophages were absent when the transplant was conducted at < 50 gestational days, implying that regulatory T cells and premature innate immunity might have contributed to the sustained engraftment. In conclusion, long-term macroscopic engraftment of primate ES cells in sheep is feasible despite the xenogeneic immunological barrier.