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A versatile in vivo platform for reversible control of transgene expression in adult tissues. 一个多功能的体内平台可逆控制转基因表达在成人组织。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2025-01-14 Epub Date: 2024-12-05 DOI: 10.1016/j.stemcr.2024.11.003
Jumpei Taguchi, Yosuke Yamada, Sho Ohta, Fumie Nakasuka, Takuya Yamamoto, Manabu Ozawa, Yasuhiro Yamada

Temporal control of transgenes has advanced biomedical interventions, including in vivo reprogramming, often utilizing the doxycycline (Dox)-mediated Tet-ON system. Here, we developed the Dox-mediated Tet-ON or complementary Tet-OFF counterpart to thoroughly investigate spatial and temporal transgene regulation in adult tissues, revealing inherent limitations and unexpected capabilities of each system. In stark contrast with the Tet-ON system, which was effective only in particular tissues and cell types, primarily epithelial cells, the Tet-OFF system proved capable of gene induction across diverse cell types. Despite the drawback of the Tet-OFF system in inducibility and tunability identified in our study, we demonstrated that use of tetracycline (Tc) effectively addresses these issues, possibly through its pharmacologic properties. Our data suggest that the Tc-mediated Tet-OFF system not only enables more versatile control of transgene expression but also offers a more biocompatible alternative for in vivo applications such as tissue regeneration and organismal rejuvenation.

转基因的时间控制具有先进的生物医学干预措施,包括体内重编程,通常利用强力霉素(Dox)介导的Tet-ON系统。在这里,我们开发了dox介导的Tet-ON或互补的Tet-OFF对应物,以彻底研究成人组织中的空间和时间转基因调控,揭示了每个系统的固有局限性和意想不到的能力。Tet-ON系统仅在特定组织和细胞类型(主要是上皮细胞)中有效,与之形成鲜明对比的是,Tet-OFF系统被证明能够在多种细胞类型中进行基因诱导。尽管在我们的研究中发现了Tet-OFF系统在诱导性和可调性方面的缺点,但我们证明了四环素(Tc)的使用可能通过其药理学特性有效地解决了这些问题。我们的数据表明,tc介导的Tet-OFF系统不仅能够更灵活地控制转基因表达,而且还为组织再生和有机体再生等体内应用提供了更生物相容性的选择。
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引用次数: 0
Engineering the 3D structure of organoids. 设计类器官的三维结构。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2025-01-14 Epub Date: 2024-12-19 DOI: 10.1016/j.stemcr.2024.11.009
Samuel P Moss, Ezgi Bakirci, Adam W Feinberg

Organoids form through the sel f-organizing capabilities of stem cells to produce a variety of differentiated cell and tissue types. Most organoid models, however, are limited in terms of the structure and function of the tissues that form, in part because it is difficult to regulate the cell type, arrangement, and cell-cell/cell-matrix interactions within these systems. In this article, we will discuss the engineering approaches to generate more complex organoids with improved function and translational relevance, as well as their advantages and disadvantages. Additionally, we will explore how biofabrication strategies can manipulate the cell composition, 3D organization, and scale-up of organoids, thus improving their utility for disease modeling, drug screening, and regenerative medicine applications.

类器官通过干细胞的自组织能力形成,产生各种分化的细胞和组织类型。然而,大多数类器官模型在形成的组织的结构和功能方面受到限制,部分原因是难以调节这些系统中的细胞类型、排列和细胞-细胞/细胞-基质相互作用。在本文中,我们将讨论产生更复杂的类器官的工程方法,这些方法具有改进的功能和翻译相关性,以及它们的优缺点。此外,我们将探索生物制造策略如何操纵细胞组成、3D组织和类器官的放大,从而提高它们在疾病建模、药物筛选和再生医学应用中的效用。
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引用次数: 0
Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels. 新生基质沉积支持合成水凝胶聚集体形成肺泡类器官。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2025-01-14 Epub Date: 2024-12-12 DOI: 10.1016/j.stemcr.2024.11.006
Madeline K Eiken, Charlie J Childs, Lindy K Brastrom, Tristan Frum, Eleanor M Plaster, Donia W Ahmed, Ryan C Spencer, Orren Shachaf, Suzanne Pfeiffer, Justin E Levine, Konstantinos-Dionysios Alysandratos, Darrell N Kotton, Jason R Spence, Claudia Loebel

Human induced pluripotent stem cell (iPSC)-derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse sarcoma-derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in synthetic hydrogels, which supports their growth. Thus, the synthetic hydrogels described here allow us to de-couple exogenous and nascent ECM to interrogate the role of ECM in organoid formation.

人类诱导多能干细胞(iPSC)衍生的肺泡器官组织已成为模拟肺泡上皮细胞稳态和疾病的系统。然而,肺泡器官组织通常是在源自小鼠肉瘤的基底膜基质 Matrigel 中生长的,这种基质对基质特性的控制很差,这促使人们开发合成水凝胶作为 Matrigel 的替代品。在这里,我们开发了一种两步培养法,即先在基于水凝胶的微孔中预聚有机体,然后将其包埋在合成水凝胶中,这种方法支持肺泡有机体的生长,同时还能对有机体和水凝胶的特性进行很好的控制。我们发现,无论是在微孔中还是嵌入合成水凝胶后,聚集的类器官都会分泌其自身的新生细胞外基质(ECM),从而支持其生长。因此,本文所述的合成水凝胶使我们能够将外源 ECM 与新生 ECM 分离,从而研究 ECM 在类器官形成过程中的作用。
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引用次数: 0
Exploiting O-GlcNAc dyshomeostasis to screen O-GlcNAc transferase intellectual disability variants. 利用O-GlcNAc失衡筛选O-GlcNAc转移酶智力残疾变异。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2025-01-14 Epub Date: 2024-12-19 DOI: 10.1016/j.stemcr.2024.11.010
Huijie Yuan, Conor W Mitchell, Andrew T Ferenbach, Maria Teresa Bonati, Agnese Feresin, Paul J Benke, Queenie K G Tan, Daan M F van Aalten

O-GlcNAcylation is an essential protein modification catalyzed by O-GlcNAc transferase (OGT). Missense variants in OGT are linked to a novel intellectual disability syndrome known as OGT congenital disorder of glycosylation (OGT-CDG). The mechanisms by which OGT missense variants lead to this heterogeneous syndrome are not understood, and no unified method exists for dissecting pathogenic from non-pathogenic variants. Here, we develop a double-fluorescence strategy in mouse embryonic stem cells to measure disruption of O-GlcNAc homeostasis by quantifying the effects of variants on endogenous OGT expression. OGT-CDG variants generally elicited a lower feedback response than wild-type and Genome Aggregation Database (gnomAD) OGT variants. This approach was then used to dissect new putative OGT-CDG variants from pathogenic background variants in other disease-associated genes. Our work enables the prediction of pathogenicity for rapidly emerging de novo OGT-CDG variants and points to reduced disruption of O-GlcNAc homeostasis as a common mechanism underpinning OGT-CDG.

O-GlcNAc酰化是由O-GlcNAc转移酶(OGT)催化的一种重要的蛋白质修饰。OGT的错义变异与一种被称为OGT先天性糖基化障碍(OGT- cdg)的新型智力残疾综合征有关。OGT错义变异体导致这种异质性综合征的机制尚不清楚,也没有统一的方法来区分致病变异体和非致病变异体。在这里,我们在小鼠胚胎干细胞中开发了一种双荧光策略,通过量化变异对内源性OGT表达的影响来测量O-GlcNAc稳态的破坏。OGT- cdg变异通常比野生型和基因组聚集数据库(gnomAD) OGT变异引起更低的反馈反应。然后使用这种方法从其他疾病相关基因的致病背景变异中解剖新的假定的OGT-CDG变异。我们的工作能够预测快速出现的新OGT-CDG变异的致病性,并指出减少对O-GlcNAc稳态的破坏是OGT-CDG的共同机制。
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引用次数: 0
Chemically defined and growth factor-free system for highly efficient endoderm induction of human pluripotent stem cells. 化学定义的无生长因子系统,用于高效的人多能干细胞内胚层诱导。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2025-01-14 Epub Date: 2024-12-26 DOI: 10.1016/j.stemcr.2024.11.012
Zhiju Zhao, Fanzhu Zeng, Yage Nie, Gang Lu, He Xu, He En, Shanshan Gu, Wai-Yee Chan, Nan Cao, Jia Wang

Definitive endoderm (DE) derived from human pluripotent stem cells (hPSCs) holds great promise for cell-based therapies and drug discovery. However, current DE differentiation methods required undefined components and/or expensive recombinant proteins, limiting their scalable manufacture and clinical use. Homogeneous DE differentiation in defined and recombinant protein-free conditions remains a major challenge. Here, by systematic optimization and high-throughput screening, we report a chemically defined, small-molecule-based defined system that contains only four components (4C), enabling highly efficient and cost-effective DE specification of hPSCs in the absence of recombinant proteins. 4C-induced DE can differentiate into functional hepatocytes, lung epithelium, and pancreatic β cells in vitro and multiple DE derivatives in vivo. Genomic accessibility analysis reveals that 4C reconfigures chromatin architecture to allow key DE transcription factor binding while identifying TEAD3 as a novel key regulator of the process. This system may facilitate mass production of DE derivatives for drug discovery, disease modeling, and cell therapy.

来自人类多能干细胞(hPSCs)的最终内胚层(DE)在基于细胞的治疗和药物发现方面具有很大的前景。然而,目前的DE分化方法需要未定义的成分和/或昂贵的重组蛋白,限制了它们的可扩展制造和临床应用。在定义蛋白和重组蛋白无条件下均质DE分化仍然是一个主要挑战。在这里,通过系统优化和高通量筛选,我们报告了一个化学定义的、基于小分子的定义系统,它只包含四个组分(4C),在没有重组蛋白的情况下,能够高效和经济地对hPSCs进行DE规范。4c诱导的DE在体外可分化为功能性肝细胞、肺上皮细胞和胰腺β细胞,在体内可分化为多种DE衍生物。基因组可及性分析显示,4C重新配置染色质结构,允许关键DE转录因子结合,同时确定TEAD3是该过程的一个新的关键调节因子。该系统可促进用于药物发现、疾病建模和细胞治疗的DE衍生物的大规模生产。
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引用次数: 0
Deep-supercooling preservation of stem cell spheroids for chondral defect repairment. 用于软骨缺损修复的干细胞球体的深度过冷保存。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2024-12-10 Epub Date: 2024-11-21 DOI: 10.1016/j.stemcr.2024.10.008
Jinbin Qiu, Bangrui Yu, Cheng Ren, Tian Wang, Guangjian Zhang, Zhe Jian, Jian Ding, Feng Xu, Haishui Huang

Versatile mesenchymal stem cells (MSCs) play an important role in tissue engineering and regenerative medicine. MSCs in 3D spheroid have shown higher secretion and differentiation functions than suspended counterparts, and, thus, in vitro cryopreservation of MSC spheroids is an indispensable technology to bridge the spatiotemporal gaps between spheroid generation and application. Traditional cryopreservation methods are inapplicable for spheroid due to severe thermal stress, toxic cryoprotectants, and ice formation. Here, we constructed and preserved human MSC (hMSC) spheroids via deep supercooling (DSC). Spheroids were DSC preserved at -12°C without ice formation for 7 days, with higher cell viability, energy level, and chondrogenic differentiation capacity than suspended hMSCs. hMSCs embedded in spheroids have close cell-cell interactions via N-cadherin to activate the AKT-cytochrome c-caspase anti-apoptotic cascade during DSC preservation. Finally, preserved hMSC spheroids were capable of chondrogenic differentiation and can be co-delivered with collagen to treat rat cartilage defects in vivo.

多功能间充质干细胞(MSCs)在组织工程和再生医学中发挥着重要作用。与悬浮的间充质干细胞相比,三维球形的间充质干细胞具有更高的分泌和分化功能,因此,间充质干细胞球体的体外冷冻保存是弥合球体生成与应用之间时空差距的一项不可或缺的技术。由于严重的热应力、有毒的冷冻保护剂和冰的形成,传统的冷冻保存方法不适用于球形干细胞。在这里,我们通过深度过冷(DSC)技术构建并保存了人间叶干细胞(hMSC)球体。与悬浮的 hMSCs 相比,球体内的 hMSCs 具有更高的细胞活力、能量水平和软骨分化能力。在 DSC 保存过程中,球体内的 hMSCs 通过 N-cadherin 与细胞紧密相互作用,激活 AKT-细胞色素 c-天冬酶抗凋亡级联。最后,被保存的hMSC球体能够进行软骨分化,并能与胶原蛋白共同输送,用于治疗体内大鼠软骨缺损。
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引用次数: 0
Interleukin-1β induces trained innate immunity in human hematopoietic progenitor cells in vitro. 白细胞介素-1β可诱导体外人类造血祖细胞产生训练有素的先天性免疫。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2024-12-10 Epub Date: 2024-11-07 DOI: 10.1016/j.stemcr.2024.09.004
Daniela Flores-Gomez, Willemijn Hobo, Diede van Ens, Elise L Kessler, Boris Novakovic, Nicolaas P M Schaap, Wim H C Rijnen, Leo A B Joosten, Mihai G Netea, Niels P Riksen, Siroon Bekkering

Innate immune cells can develop a long-lasting hyperresponsive phenotype, termed trained immunity, mediated by epigenetic and metabolic reprogramming. In mice, exposure to Bacille Calmette-Guérin (BCG), β-glucan, or Western diet induces trained immunity by reprogramming hematopoietic progenitor cells (HPCs), through interleukin-1β (IL-1β) signaling in the bone marrow (BM). We investigated whether IL-1β induces trained immunity in primary human BM-derived HPCs in vitro. We exposed human BM-derived HPCs to IL-1β for 4 h. HPCs were expanded and differentiated into monocytes followed by functional and transcriptomic characterization. IL-1β-exposed HPCs showed higher granulocyte-macrophage colony-forming units. The monocyte offspring produced more tumor necrosis factor (TNF) and IL-1β after restimulation with lipopolysaccharide (LPS) and Pam3Cys and is metabolically more active. Transcriptomic analysis showed upregulation of key atherogenic and inflammatory pathways. In conclusion, brief exposure of human BM-derived HPCs to IL-1β in vitro induces a trained immunity phenotype.

先天性免疫细胞可在表观遗传和代谢重编程的介导下,形成一种持久的高反应表型,即训练有素的免疫力。在小鼠体内,暴露于卡介苗(Bacille Calmette-Guérin,BCG)、β-葡聚糖或西方饮食可通过骨髓(BM)中的白细胞介素-1β(IL-1β)信号传导重编程造血祖细胞(HPCs),从而诱导训练有素的免疫。我们研究了IL-1β是否能在体外诱导原代人BM衍生的HPC产生训练有素的免疫力。我们将人骨髓来源的 HPCs 暴露于 IL-1β 4 小时。HPCs 扩增并分化成单核细胞,然后进行功能和转录组学表征。暴露于 IL-1β 的 HPCs 表现出更高的粒细胞-巨噬细胞集落形成单位。经脂多糖(LPS)和 Pam3Cys 再刺激后,单核细胞后代产生更多肿瘤坏死因子(TNF)和 IL-1β,而且代谢更活跃。转录组分析表明,关键的致动脉粥样硬化和炎症通路上调。总之,人血浆来源的 HPCs 在体外短暂暴露于 IL-1β 可诱导训练有素的免疫表型。
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引用次数: 0
Derivation of transplantable human thyroid follicular epithelial cells from induced pluripotent stem cells. 从诱导多能干细胞中衍生出可移植的人类甲状腺滤泡上皮细胞。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2024-12-10 Epub Date: 2024-11-07 DOI: 10.1016/j.stemcr.2024.10.004
Hendrik J Undeutsch, Alberto Posabella, Andrea B Alber, Pushpinder S Bawa, Carlos Villacorta-Martin, Feiya Wang, Laertis Ikonomou, Darrell N Kotton, Anthony N Hollenberg

The production of mature functioning thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) is critical for potential novel therapeutic approaches to post-surgical and congenital hypothyroidism. To accomplish this, we developed a novel human iPSC line that expresses fluorophores targeted to the NKX2-1 and PAX8 loci, allowing for the identification and purification of cells destined to become TFCs. Optimizing a sequence of defined, serum-free media to promote stepwise developmental directed differentiation, we found that bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) stimulated lineage specification into TFCs from multiple iPSC lines. Single-cell RNA sequencing demonstrated that BMP4 withdrawal after lineage specification promoted TFC maturation, with mature TFCs representing the majority of cells present within 1 month. After xenotransplantation into athyreotic immunodeficient mice, engrafted cells exhibited thyroid follicular organization with thyroglobulin protein detected in the lumens of NKX2-1-positive follicles. While our iPSC-derived TFCs presented durable expression of thyroid-specific proteins, they were unable to rescue hypothyroidism in vivo.

从人类诱导多能干细胞(iPSC)中培育出功能成熟的甲状腺滤泡细胞(TFC),对于手术后和先天性甲状腺功能减退症的潜在新型治疗方法至关重要。为了实现这一目标,我们开发了一种新型人类 iPSC 细胞系,该细胞系能表达针对 NKX2-1 和 PAX8 基因座的荧光团,从而识别和纯化注定要成为 TFC 的细胞。我们发现,骨形态发生蛋白4(BMP4)和成纤维细胞生长因子2(FGF2)能刺激多个iPSC品系的TFC细胞系分化。单细胞RNA测序表明,在品系分化后撤除BMP4可促进TFC的成熟,1个月内成熟的TFC占细胞总数的大多数。异种移植到无甲状腺免疫缺陷小鼠体内后,移植细胞表现出甲状腺滤泡组织,在NKX2-1阳性滤泡的管腔中检测到甲状腺球蛋白蛋白。虽然我们的iPSC衍生TFCs能持久表达甲状腺特异性蛋白,但它们无法挽救体内甲状腺功能减退症。
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引用次数: 0
Bone morphogenetic protein 4 induces hematopoietic stem cell development from murine hemogenic endothelial cells in culture. 骨形态发生蛋白 4 可诱导小鼠造血内皮细胞的造血干细胞发育。
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2024-12-10 Epub Date: 2024-11-14 DOI: 10.1016/j.stemcr.2024.10.005
Mariko Tsuruda, Saori Morino-Koga, Xueyu Zhao, Shingo Usuki, Kei-Ichiro Yasunaga, Tomomasa Yokomizo, Ryuichi Nishinakamura, Toshio Suda, Minetaro Ogawa

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) during mouse embryogenesis. Understanding the signaling molecules required for HSC development is crucial for the in vitro derivation of HSCs. We previously induced HSCs from embryonic HECs, isolated at embryonic day 10.5 (E10.5), in serum-free culture conditions with stem cell factor, thrombopoietin, and an endothelial feeder layer. Here, we aimed to elucidate signal requirements for inducing HSCs from earlier-stage HECs. Single-cell RNA sequencing (RNA-seq) analysis detected bone morphogenetic protein (BMP) signaling activation in E9.5 HECs. Adding BMP4 to the culture conditions led to the induction of HSCs from E9.5 HECs. Furthermore, isolating BMP4 receptor-expressing HECs from E9.5 embryos enriched progenitors with HSC-forming ability. This study identified BMP4 as an essential factor promoting the differentiation of early HECs into HSCs, opening up new possibilities for the in vitro derivation of HSCs.

造血干细胞(HSCs)是在小鼠胚胎发育过程中由造血内皮细胞(HECs)发育而来的。了解造血干细胞发育所需的信号分子对体外衍生造血干细胞至关重要。我们以前曾在无血清培养条件下,用干细胞因子、血小板生成素和内皮馈源层从胚胎10.5天(E10.5)分离的胚胎HECs中诱导出造血干细胞。在此,我们旨在阐明从早期HECs诱导造血干细胞的信号要求。单细胞 RNA 测序(RNA-seq)分析检测到 E9.5 HECs 中的骨形态发生蛋白(BMP)信号激活。在培养条件中加入BMP4可从E9.5 HECs中诱导出造血干细胞。此外,从E9.5胚胎中分离出表达BMP4受体的HECs还能富集具有造血干细胞形成能力的祖细胞。这项研究确定了 BMP4 是促进早期 HECs 向 HSCs 分化的重要因子,为体外衍生 HSCs 开辟了新的可能性。
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引用次数: 0
Amendments to ASRM: Can we move away from a "Therapeutic Haven"? ASRM 修正案:我们能否放弃 "治疗天堂"?
IF 5.9 2区 医学 Q1 CELL & TISSUE ENGINEERING Pub Date : 2024-12-10 Epub Date: 2024-11-14 DOI: 10.1016/j.stemcr.2024.10.006
Tsunakuni Ikka, Taichi Hatta, Misao Fujita

The key amendment to the Act on the Safety of Regenerative Medicine in June 2024 is regarding on-site inspections and the criteria for disqualifying the Certified Special Committees for Regenerative Medicine and Certified Committees for Regenerative Medicine. Appropriate regulations are needed after the legal amendment to stop the widespread use of unproven interventions and move away from the concept of a "Therapeutic Haven."

2024 年 6 月对《再生医学安全法》的主要修订涉及现场检查以及取消再生医学认证特别委员会和再生医学认证委员会资格的标准。法律修订后需要制定适当的法规,以阻止未经证实的干预措施的广泛使用,并摒弃 "治疗天堂 "的概念。
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引用次数: 0
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Stem Cell Reports
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