Generation of induced pluripotent stem cells from the Asian bats.

Preservation of native Korean bats is crucial for maintaining ecological balance, as they play a vital role in insect control, pollination, and seed dispersal within their ecosystems. The present study details the establishment of bat induced pluripotent stem cells (BatiPSCs) from two Asian and Korean bats (Hypsugo alaschanicus and Pipistrellus abramus) using the Sendai Reprogramming Kit. Colonies of BatiPSCs, exhibiting distinctive features, were manually selected and expanded following successful transfection. Characterization of BatiPSCs revealed the expression of pluripotency markers, such as Octamer-binding transcription factor 4 (Oct4), SRY (sex-determining region Y)-box 2 and Nanog, with notably increased Oct4 levels and reduced Myc proto-oncogene expression compared with those noted in other induced pluripotent stem cell sources. BatiPSCs displayed positive staining for alkaline phosphatase and demonstrated the ability to form embryoid bodies, while also inducing teratomas in non-immune nude mice. Additionally, green fluorescent protein (GFP)-expressing BatiPSCs were generated and used for chimeric mouse production, with slight GFP signals detected in the neck region of the resulting mouse foetuses. These findings demonstrate the successful generation and characterization of BatiPSCs, emphasizing their potential applications in chimeric animal models, and the protection of endangered bat species.

Applications of CRISPR technologies to the development of gene and cell therapy.

Advancements in gene and cell therapy have resulted in novel therapeutics for diseases previously considered incurable or challenging to treat. Among the various contributing technologies, genome editing stands out as one of the most crucial for the progress in gene and cell therapy. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the subsequent evolution of genetic engineering technology have markedly expanded the field of target-specific gene editing. Originally studied in the immune systems of bacteria and archaea, the CRISPR system has demonstrated wide applicability to effective genome editing of various biological systems including human cells. The development of CRISPR-based base editing has enabled directional cytosine-tothymine and adenine-to-guanine substitutions of select DNA bases at the target locus. Subsequent advances in prime editing further elevated the flexibility of the edit multiple consecutive bases to desired sequences. The recent CRISPR technologies also have been actively utilized for the development of in vivo and ex vivo gene and cell therapies. We anticipate that the medical applications of CRISPR will rapidly progress to provide unprecedented possibilities to develop novel therapeutics towards various diseases. [BMB Reports 2024; 57(1): 2-11].

Morphologically and karyotypically atypical cells of 'normal' human bronchial epithelial cell line (Beas-2B).

Beas-2B is an adenovirus 12-SV40-transfected cell line of "normal" human bronchial epithelial cells. This cell line was able to replace normal human bronchial epithelial cells, which are currently unavailable, and served as a model for related studies in numerous toxicology and cancer transformation experiments. In any experiment involving toxins or carcinogens, the basic morphology of Beas-2B should be well characterized prior to exposure, but this has never been properly reported. In this study, atypical cells of the Beas-2B cell line in early passage culture were observed using light and electron microscopy, and the cells were further investigated for abnormal karyotypes by flow cytometry. This Beas-2B cell line could be morphologically categorized into two cell types, A and B. Type A contains a large nucleus and abundant cytoplasm (type A > 95%) and type B contains a small nucleus with dense and scarce cytoplasm (type B < 5%). Both atypical cell types had atypical and multilobed/multinucleated cells, including a high percentage (>30%) of mitotic figures, and were Ki-67 positive (100%). Karyotyping also revealed that 40.4% of the cells had atypical karyotyped chromosomes. In light of these findings, this cell line is no longer a "normal" cell, and experiments performed using this cell line can be questioned for non-default results. Experimenters should consider this error in future experiments.

Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells.

Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

The Fruiting Body Formation of Oudemansiella radicata in the Sawdust of Oak (Quercus variabilis) Mixed with Rice Bran.

To screen additives and their mixed ratio suitable for the mycelial growth and fruiting body formation of Oudemansiella radicata in the oak sawdust, additives such as rice bran, fermented soybean powder and wheat bran were used. Generally, the mycelial growth of O. radicata has been stable on oak sawdust mixed with rice bran of 5~20%. In case that O. radicata was cultured for about 30 days at 22 ± 1℃ under the illumination (350 lux) of 12 hours and moisture condition of 90 ± 5%, the primordia have been formed gradually from red-brown crusts covering the surface of oak sawdust media. Based on the experimental results from 9 strains of O. radicata, fruiting bodies were produced widely on oak sawdust medium mixed with rice bran of 5 to 30%. Even though fruiting bodies of O. radicata have been produced well on oak sawdust media mixed with rice bran, fruiting bodies of O. radicata were produced intensively on oak sawdust media mixed with rice bran of 10%. Therefore, this result will provide a basic information for commercial production of fruiting body of wild O. radicata. This result is the first report associated with an artificial fruiting body formation of O. radicata in Korea.

The Fruiting Body Formation of Armillaria mellea on Oak Sawdust Medium Covered with Ground Raw Carrots.

To produce an artificial fruiting body of Armillaria mellea on the oak sawdust medium, seven strains of A. mellea were used. The top surface of oak sawdust medium covered with ground raw carrot was inoculated with each of 7 strains and cultured for 30 days at 25℃ in the dark condition until the mycelia of A. mellea completely colonized the medium from top to bottom. Then, the mycelia which were fully covered on the top surface of the medium were scratched slightly with a spatula and filled with tap water for 3 hours. To induce the primordial formation, the 7 strains of A. mellea were transferred to the growth chamber under the illumination (350 lux) of 12 hours and relative humidity of 85 ± 5% in a day and then cultured at 16 ± 1℃. Only A. mellea IUM 949 could form primordia on the sawdust medium, but the other strains did not make primordia at the same condition. The primordia of A. mellea IUM 949 were formed 10 days after complete colonization of the medium and the fruiting bodies were produced 7 days after a primordial formation. The experimental results suggested that IUM 949 strain might be a good candidate for mass production of fruiting bodies of A. mellea.