Microbiota-derived butyrate restricts tuft cell differentiation via histone deacetylase 3 to modulate intestinal type 2 immunity.

Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.

The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

A highly reproducible and efficient method for retinal organoid differentiation from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Gluten dependent activation of CD4+ T cells by MHC class II-expressing epithelium.

BACKGROUND AND AIMS: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T cell interactions in organoid monolayers expressing human MHC class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD. METHODS: Epithelial MHC class II (MHCII) was determined in active and treated CeD, and in non-immunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without IFN-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T cell co-cultures were incubated with gluten, pre-digested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants. RESULTS: Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer-T cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T cell activation markers, and the release of IL-2, IFN-γ, and IL-15 in co-culture supernatants. Gluten metabolized by P. aeruginosa, but not the lasB mutant, enhanced CD4+ T cell proliferation and activation. CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten pre-digestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD patients.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Past and future of alveolar organoids for lung regenerative medicine.

The lung is regarded as having limited regenerative capacity, and there are few treatment options for refractory lung diseases, such as interstitial pneumonia. Lung transplantation is the final option available in some scenarios. Research in this area has been slow owing to the complex structure of the lung for efficient gas exchange between the alveolar spaces and capillaries as well as the difficulty in obtaining specimens from patients with progressive lung disease. However, basic research over the past decade in the field of mouse and human embryology using genetic lineage tracing techniques and stem cell biology using primary and pluripotent stem cell-derived alveolar organoids has begun to clarify the tissue response in various intractable lung diseases and the mechanisms underlying remodeling. Advancement in this area may expand potential therapeutic targets for alveolar regeneration, providing alternatives to lung transplantation, and contribute to the development of effective therapeutic methods that activate or repopulate stem cells in the lung. In this review, we cover research focused on alveolar epithelial cells and discuss methods expected to regenerate lungs that are damaged by diseases.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Low-intensity ultrasound ameliorates brain organoid integration and rescues microcephaly deficits.

Human brain organoids represent a remarkable platform for modeling neurological disorders and a promising brain repair approach. However, the effects of physical stimulation on their development and integration remain unclear. Here, we report that low-intensity ultrasound significantly increases neural progenitor cell proliferation and neuronal maturation in cortical organoids. Histological assays and single-cell gene expression analyses reveal that low-intensity ultrasound improves the neural development in cortical organoids. Following organoid grafts transplantation into the injured somatosensory cortices of adult mice, longitudinal electrophysiological recordings and histological assays reveal that ultrasound-treated organoid grafts undergo advanced maturation. They also exhibit enhanced pain-related gamma-band activity and more disseminated projections into the host brain than the untreated groups. Finally, low-intensity ultrasound ameliorates neuropathological deficits in a microcephaly brain organoid model. Hence, low-intensity ultrasound stimulation advances the development and integration of brain organoids, providing a strategy for treating neurodevelopmental disorders and repairing cortical damage.

Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

MSCs alleviate LPS-induced acute lung injury by inhibiting the proinflammatory function of macrophages in mouse lung organoid-macrophage model.

Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.

PHLDA1-PRDM1 mediates the effect of lentiviral vectors on fate-determination of human retinal progenitor cells.

Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis.

Bioengineered MSCCxcr2 transdifferentiated keratinocyte-like cell-derived organoid potentiates skin regeneration through ERK1/2 and STAT3 signaling in diabetic wound.

Skin regeneration is severely compromised in diabetic foot ulcers. Allogeneic mesenchymal stem cell (MSC) transplantation is limited due to the poor engraftment, mitogenic, and differentiation potential in the harsh wound microenvironment. Thus, to improve the efficacy of cell therapy, the chemokine receptor Cxcr2 was overexpressed in MSCs (MSCCxcr2). CXCL2/CXCR2 axis induction led to the enhanced proliferation of MSCs through the activation of STAT3 and ERK1/2 signaling. Transcriptional upregulation of FGFR2IIIb (KGF Receptor) promoter by the activated STAT3 and ERK1/2 suggested trans-differentiation of MSCs into keratinocytes. These stable MSCCxcr2 in 2D and 3D (spheroid) cell cultures efficiently transdifferentiated into keratinocyte-like cells (KLCs). An in vivo therapeutic potential of MSCCxcr2 transplantation and its keratinocyte-specific cell fate was observed by accelerated skin tissue regeneration in an excisional splinting wound healing murine model of streptozotocin-induced type 1 diabetes. Finally, 3D skin organoids generated using MSCCxcr2-derived KLCs upon grafting in a relatively avascular and non-healing wounds of type 2 diabetic db/db transgenic old mice resulted in a significant enhancement in the rate of wound closure by increased epithelialization (epidermal layer) and endothelialization (dermal layer). Our findings emphasize the therapeutic role of the CXCL2/CXCR2 axis in inducing trans-differentiation of the MSCs toward KLCs through the activation of ERK1/2 and STAT3 signaling and enhanced skin regeneration potential of 3D organoids grafting in chronic diabetic wounds.

Pax6 isoforms shape eye development: Insights from developmental stages and organoid models.

Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.

Novel Organoid Culture System for Improved Safety Assessment of Nanomaterials.

Over the past few decades, the increased application of nanomaterials has raised questions regarding their safety and possible toxic effects. Organoids have been suggested as promising tools, offering efficient assays for nanomaterial-induced toxicity evaluation. However, organoid systems have some limitations, such as size heterogeneity and poor penetration of nanoparticles because of the extracellular matrix, which is necessary for organoid culture. Here, we developed a novel system for the improved safety assessment of nanomaterials by establishing a 3D floating organoid paradigm. In addition to overcoming the limitations of two-dimensional systems including the lack of in vitro-in vivo cross-talk, our method provides multiple benefits as compared with conventional organoid systems that rely on an extracellular matrix for culture. Organoids cultured using our method exhibited relatively uniform sizing and structural integrity and were more conducive to the internalization of nanoparticles. Our floating culture system will accelerate the research and development of safe nanomaterials.

Using Organoids to Tap Mammary Gland Diversity for Novel Insight.

This article offers a comprehensive perspective on the transformative role of organoid technology on mammary gland biology research across a diverse array of mammalian species.The mammary gland's unique development and regenerative capabilities render this organ an ideal model for studying developmental evolution, stem cell behavior, and regenerative processes. The discussion extends to the use of cross-species mammary organoids to address key biological inquiries in evolution, tissue regeneration, cancer research, and lactation, highlighting the limitations of traditional mouse models and the benefits of incorporating a more diverse range of animal models.Advances in organoid biology have been critical in overcoming ethical and practical constraints of in-vivo studies, especially in human research. The generation of human and mouse mammary organoids that faithfully recapitulate in-vivo tissues marks a significant stride in this field. Parallel capabilities are now emerging for other mammals, as well.Utilizing mammary organoids from various species has the potential to make invaluable contributions to our understanding of mammary gland biology, with implications for regenerative medicine, cancer research, and lactation studies, thereby contributing to advancements in human health, agriculture, and nutrition science.

Lipid Deficiency Contributes to Impaired Alveolar Progenitor Cell Function in Aging and Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2s) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of patients with IPF and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single-cell RNA sequencing, we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed downregulation of genes related to lipid biosynthesis and fatty acid ß-oxidation in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs compared with the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, old mouse lungs using immunofluorescence staining and flow cytometry. Futhermore, we show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and PPARγ (peroxisome proliferator activated receptor γ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in three-dimensional organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of SFTPC in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured old mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids.

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.

Transplantation of beige adipose organoids fabricated using adipose acellular matrix hydrogel improves metabolic dysfunction in high-fat diet-induced obesity and type 2 diabetes mice.

Transplantation of brown adipose tissue (BAT) is a promising approach for treating obesity and metabolic disorders. However, obtaining sufficient amounts of functional BAT or brown adipocytes for transplantation remains a major challenge. In this study, we developed a hydrogel that combining adipose acellular matrix (AAM) and GelMA and HAMA that can be adjusted for stiffness by modulating the duration of light-crosslinking. We used human white adipose tissue-derived microvascular fragments to create beige adipose organoids (BAO) that were encapsulated in either a soft or stiff AAM hydrogel. We found that BAOs cultivated in AAM hydrogels with high stiffness demonstrated increased metabolic activity and upregulation of thermogenesis-related genes. When transplanted into obese and type 2 diabetes mice, the HFD + BAO group showed sustained improvements in metabolic rate, resulting in significant weight loss and decreased blood glucose levels. Furthermore, the mice showed a marked reduction in nonalcoholic liver steatosis, indicating improved liver function. In contrast, transplantation of 2D-cultured beige adipocytes failed to produce these beneficial effects. Our findings demonstrate the feasibility of fabricating beige adipose organoids in vitro and administering them by injection, which may represent a promising therapeutic approach for obesity and diabetes.