Satoshi Watanabe

Background: Dental pulp (DP) is a connective tissue composed of various cell types, including fibroblasts, neurons, adipocytes, endothelial cells, and odontoblasts. It contains a rich supply of pluripotent stem cells, making it an important resource for cell-based regenerative medicine. However, current stem cell collection methods rely heavily on the enzymatic digestion of dissected DP tissue to isolate and propagate primary cells, which often results in low recovery rates and reduced cell survival, particularly from deciduous teeth. Methods: We developed a novel and efficient method to obtain a sufficient number of cells through a one-step cultivation process of isolated DP. After the brief digestion of DP with proteolytic enzymes, it was scratched onto a culture dish and cultured in a suitable medium. By day 2, the cells began to spread radially from DP, and by day 10, they reached a semi-confluent state. Cells harvested through trypsinization consistently yielded over 1 million cells, and after re-cultivation, the cells could be propagated for more than ten passages. Results: The proliferative and differentiation capacities of the cells after the 10th passage were comparable to those from the first passage. The cells expressed alkaline phosphatase as an undifferentiation marker. Similarly, they also maintained the constitutive expression of stem cell-specific markers and differentiation-related markers, even after the 10th passage. Conclusions: This method, termed “scratch-based isolation of primary cells from human dental pulps (SCIP)”, enables the efficient isolation of a large number of DP cells with minimal equipment and operator variability, while preserving cell integrity. Its simplicity, high success rate, and adaptability for patients with genetic diseases make it a valuable tool for regenerative medicine research and clinical applications.

Gene-engineered animals created using gene-targeting technology have long been recognized as beneficial, valid, and valuable tools for exploring the function of a gene of interest, at least in early 2013. This approach, however, suffers from laborious and time-consuming tasks, such as the production of successfully targeted embryonic stem (ES) cells, their characterization, production of chimeric blastocysts carrying these gene-modified ES cells, and transplantation of those manipulated blastocysts to the recipient (pseudopregnant) females to deliver chimeric mice. Since the appearance of genome editing technology, which is now exemplified by the CRISPR/Cas9 system, in late 2013, significant advances have been made in the generation of genome-edited animals through pronuclear microinjection (MI) of genome-editing components into fertilized eggs (zygotes) or electroporation (EP) of zygotes in the presence of these reagents. However, these procedures require the transfer of genome-edited embryos into the reproductive tracts of recipient females for further development. Genome editing via oviductal nucleic acids delivery (GONAD) and its modified version, called "improved GONAD (i-GONAD)," were developed as an alternative to the MI- or EP-based genome-edited animal production and now recognized to be very convenient and straightforward as genome editing can only be performed in vivo (within the oviductal lumen where fertilized embryos exist). This system also enables the simultaneous transfection of epithelial cells lining the oviductal lumen. In this review, we summarize the recent advances in GONAD/i-GONAD and their derivatives and discuss the potential of these technologies to study various biological systems related to female reproduction.

Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called “in vivo somatic cell genome editing”. This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.

The efficient production of transgenic (Tg) piglets has remained a challenge in the field of domestic animal studies. Unlike mice, the pronuclei of pig zygotes cannot be easily studied because of the abundance of lipid droplets. Therefore, the zygotes must be briefly centrifuged before pronuclear injection (PNI) to move the lipid droplets to the periphery of the zygote for PNI-mediated production of Tg piglets. However, this procedure is temporal because lipid droplets return to the original space during PNI, hampering the consecutive PNI. Cytoplasmic injection (CPI) of nucleic acids is comparatively simple than PNI because CPI does not require such pre-centrifugation. Unfortunately, CPI using purified DNA fragments is inadequate for creating Tg piglets because it is challenging to integrate nucleic acids into the host genome. PiggyBac (PB), one of the transposons, is a valuable tool enabling efficient chromosomal integration of a transgene. The PB-mediated gene transfer requires two components, namely, transposase and transposons harboring the gene of interest (GOI) flanked by PB acceptor sites. We speculate that the CPI of transposase mRNA and transposons could accelerate the chromosomal integration of the GOI in pig zygotes. To prove this hypothesis, we performed CPI using transposase mRNA (super PB transposase mRNA) + transposon DNA carrying the enhanced green fluorescent protein (EGFP) cDNA (referred to as “pT-EGFP”), transposase expression plasmid DNA (referred to as “pTrans”) + pT-EGFP, pT-EGFP alone, or non-transposon EGFP expression plasmid DNA using porcine parthenotes. Consequently, 50% (2/4 tested) of green-fluorescent embryos exhibited chromosomal integration of GOI. In contrast, green-fluorescent embryos derived from CPI with pTrans + pT-EGFP or pT-EGFP alone did not show chromosomal integration. We used mRNA for super PB transposase, which is an engineered hyperactive version of the wild-type PB transposase. To conclude, the PB system, based on the CPI of super PB transposase mRNA + transposon DNA, could be useful for producing Tg porcine parthenotes.

Adipose tissue is distributed throughout the body as fat depots. The amount of adipose tissue increases with age. In mice, epididymal fat depots in males and gonadal fat depots in females are associated with the reproductive system. Regarding fat depots in females, the adipose tissue under the skin can be easily exposed via surgery when the ovary, oviduct, and uterus are pulled out and exposed. As handling adipose tissue is relatively easy, adipocytes might be good targets for genetic manipulation (including gene delivery to the adipose cells). To examine this possibility, we injected 1 μL of dye (e.g., trypan blue or India ink) into the gonadal fat depots of female mice using a breath-controlled micropipette under a dissecting microscope. The injected dye remained at the injection site for at least one day. The injection of piggyBac (PB) transposons containing an enhanced green fluorescent protein (EGFP)-expressing unit and subsequent in vivo electroporation (EP) at the injection site resulted in the successful transfection of adipocytes. The introduction of the PB transposons caused chromosomal integration of the gene of interest. The introduction of a vector containing an octamer-binding transcription factor-3/4 promoter-directed EGFP cDNA expression unit helped to identify stem-like cells. These results supported the feasibility of our EP-based non-viral gene delivery system to transfect murine adipocytes in vivo. Using this approach, several applications such as the local production of therapeutically useful proteins, plasmid-based vaccinations, and the acquisition of immortalized adipose-derived stem cells might be possible.

Although the aging process expands the adipose tissue habitation in mice and due to its close association with the female reproductive system, it can be easily exposed surgically under anesthesia when reproductive organs (including ovary, oviduct, and part of the uterus) are pulled and exposed onto the dorsal skin. This study aimed to consider the suitability of adipose tissue as a target for manipulation, particularly for the grafting of cells or small-sized tissue sections due to its ease of handling. Subsequently, 1-2 mL trypan blue injections were administered to the tissues using a breath-controlled micropipette under a dissecting microscope for evaluating the adipose tissue’s potential as a suitable grafting material. It was observed that the injected dye remained at the injection site for at least one day after injecting B16 mouse melanoma and P19 embryonal carcinoma cells. It resulted in the generation of solid tumors surrounding the ovary, oviduct, or uterus with 100% efficiency, as reported by an inspection one and a half months after the injections. When the grafting procedure was carried out for one-fourth of the juvenile pancreas (aged 15 days), an enlarged pancreas with normal morphological configuration (including the formation of insulin-synthesizing cells) was generated, which was observed by an inspection one month after the injections, thus successfully validating our approach for adipose tissue manipulation and furthermore, naming this novel technology as “intra-adipose introduction of cells and tissues”.

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. piggyBac (PB), one of three transposons (PB, Sleeping Beauty (SB), and Tol2) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.

Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.