International Journal of Molecular Sciences

Research

16 pages, 17537 KiB

Open AccessArticle

Exosomes from Human iPSC-Derived Retinal Organoids Enhance Corneal Epithelial Wound Healing

by Si Hyung Lee, Jung Woo Han, Jin Young Yang, Jungmook Lyu, Hyo Song Park, Ji Hong Bang, Yeji Kim, Hun Soo Chang and Tae Kwann Park

Int. J. Mol. Sci. 2024, 25(16), 8925; https://doi.org/10.3390/ijms25168925 - 16 Aug 2024

Viewed by 4268

Abstract

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by [...] Read more.

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2′-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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21 pages, 12109 KiB

Open AccessArticle

Early Developmental Characteristics and Features of a Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

by Jung Woo Han, Hun Soo Chang, Sung Chul Park, Jin Young Yang, Ye Ji Kim, Jin Ha Kim, Hyo Song Park, Han Jeong, Junwon Lee, Chang Ki Yoon, Hyung Gon Yu, Se Joon Woo, Jungmook Lyu and Tae Kwann Park

Int. J. Mol. Sci. 2024, 25(15), 8203; https://doi.org/10.3390/ijms25158203 - 27 Jul 2024

Cited by 2 | Viewed by 945

Abstract

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal degeneration affecting young males caused by mutations in the retinoschisin (RS1) gene. We generated human induced pluripotent stem cells (hiPSCs) from XLRS patients and established three-dimensional retinal organoids (ROs) for disease investigation. This [...] Read more.

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal degeneration affecting young males caused by mutations in the retinoschisin (RS1) gene. We generated human induced pluripotent stem cells (hiPSCs) from XLRS patients and established three-dimensional retinal organoids (ROs) for disease investigation. This disease model recapitulates the characteristics of XLRS, exhibiting defects in RS1 protein production and photoreceptor cell development. XLRS ROs also revealed dysregulation of Na/K-ATPase due to RS1 deficiency and increased ERK signaling pathway activity. Transcriptomic analyses of XLRS ROs showed decreased expression of retinal cells, particularly photoreceptor cells. Furthermore, relevant recovery of the XLRS phenotype was observed when co-cultured with control ROs derived from healthy subject during the early stages of differentiation. In conclusion, our in vitro XLRS RO model presents a valuable tool for elucidating the pathophysiological mechanisms underlying XLRS, offering insights into disease progression. Additionally, this model serves as a robust platform for the development and optimization of targeted therapeutic strategies, potentially improving treatment outcomes for patients with XLRS. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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15 pages, 5974 KiB

Open AccessArticle

A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease

by Caren Doueiry, Christiana S. Kappler, Carla Martinez-Morant and Stephen A. Duncan

Int. J. Mol. Sci. 2024, 25(13), 7277; https://doi.org/10.3390/ijms25137277 - 2 Jul 2024

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Abstract

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant [...] Read more.

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3⁻^/⁻ and PNPLA3^I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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13 pages, 3134 KiB

Open AccessArticle

Primitive and Definitive Neural Precursor Cells Are Present in Human Cerebral Organoids

by Rehnuma Islam, Humna Noman, Ashkan Azimi, Ricky Siu, Vorapin Chinchalongporn, Carol Schuurmans and Cindi M. Morshead

Int. J. Mol. Sci. 2024, 25(12), 6549; https://doi.org/10.3390/ijms25126549 - 14 Jun 2024

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Abstract

Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the [...] Read more.

Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the NSC lineage giving rise to dNSCs in the embryonic and adult mouse brain. pNSCs are quiescent under baseline conditions and can be activated upon injury. Herein, we asked whether human pNSCs and dNSCs can be isolated during the maturation of human cerebral organoids (COs) and activated by drugs known to regulate mouse NSC behavior. We demonstrate that self-renewing, multipotent pNSC and dNSC populations are present in human COs and express genes previously characterized in mouse NSCs. The drug NWL283, an inhibitor of apoptosis, reduced cell death in COs but did not improve NSC survival. Metformin, a drug used to treat type II diabetes that is known to promote NSC activation in mice, was found to expand human NSC pools. Together, these findings are the first to identify and characterize human pNSCs, advancing our understanding of the human NSC lineage and highlighting drugs that enhance their activity. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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11 pages, 4733 KiB

Open AccessArticle

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation

by Yuka Inage, Koki Fujimori, Masaki Takasu, Kenji Matsui, Yoshitaka Kinoshita, Keita Morimoto, Nagisa Koda, Shutaro Yamamoto, Kentaro Shimada, Takashi Yokoo and Eiji Kobayashi

Int. J. Mol. Sci. 2024, 25(9), 4793; https://doi.org/10.3390/ijms25094793 - 27 Apr 2024

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Abstract

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by [...] Read more.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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14 pages, 1536 KiB

Open AccessArticle

Characterization of 3D Organotypic Culture of Mouse Adipose-Derived Stem Cells

by Tae Gen Son, Yoojin Seo, Won-Tae Kim, Meesun Kim, Seon Jeong Choi, Si Ho Choi, Byung-Jun Sung, Jae-Seok Min, Eon Chul Han and Hyung-Sik Kim

Int. J. Mol. Sci. 2024, 25(7), 3931; https://doi.org/10.3390/ijms25073931 - 1 Apr 2024

Viewed by 1388

Abstract

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their [...] Read more.

Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal–epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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Review

Jump to: Research, Other

28 pages, 2947 KiB

Open AccessReview

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm

by Vojtech Gabriel, Christopher Zdyrski, Dipak K. Sahoo, Abigail Ralston, Hannah Wickham, Agnes Bourgois-Mochel, Basant Ahmed, Maria M. Merodio, Karel Paukner, Pablo Piñeyro, Jamie Kopper, Eric W. Rowe, Jodi D. Smith, David Meyerholz, Amir Kol, Austin Viall, Mohamed Elbadawy, Jonathan P. Mochel and Karin Allenspach

Int. J. Mol. Sci. 2024, 25(2), 701; https://doi.org/10.3390/ijms25020701 - 5 Jan 2024

Cited by 2 | Viewed by 3140

Abstract

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that [...] Read more.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ’s epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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Jump to: Research, Review

14 pages, 1835 KiB

Open AccessProtocol

An Adaptable Protocol to Generate a Murine Enteroid–Macrophage Co-Culture System

by Viktoria Hentschel, Deepalakshmi Govindarajan, Thomas Seufferlein and Milena Armacki

Int. J. Mol. Sci. 2024, 25(14), 7944; https://doi.org/10.3390/ijms25147944 - 20 Jul 2024

Viewed by 1141

Abstract

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step [...] Read more.

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid–macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract. Full article

(This article belongs to the Special Issue Recent Research in Stem Cells to Organoids)

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