Organoids are novel in vitro cell culture models that enable stem cells (including pluripotent stem cells and adult stem cells) to grow and undergo self-organization within a three-dimensional microenvironment during the process of differentiation into target tissues. Such miniature structures not only recapitulate the histological and genetic characteristics of organs in vivo, but also form tissues with the capacity for self-renewal and further differentiation. Recent advances in biomaterial technology, particularly hydrogels, have provided opportunities to improve organoid cultures; by closely integrating the mechanical and chemical properties of the extracellular matrix microenvironment, with novel synthetic materials and stem cell biology. This systematic review critically examines recent advances in various strategies and techniques utilized for stem-cell-derived organoid culture, with particular emphasis on the application potential of hydrogel technology in organoid culture. We hope this will give a better understanding of organoid cultures for modelling diseases and tissue engineering applications.

Understanding mechanisms underlying the heterogeneity of multipotent stem cells offers invaluable insights into biogenesis and tissue development. Extracellular matrix (ECM) stiffness has been acknowledged as a crucial factor regulating stem cell fate. However, how cells sense stiffness cues and adapt their metabolism activity is still unknown. Here we report the novel role of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) in enhancing osteogenesis in 3D ECM via glycolysis. We experimentally mimicked the physical characteristics of 3D trabeculae network of normal and osteoporotic bone with different microstructure and stiffness, observing that PCK2 promotes osteogenesis in 3D ECM with tunable stiffness in vitro and in vivo. Mechanistically, PCK2 enhances the rate-limiting metabolic enzyme pallet isoform phosphofructokinase (PFKP) in 3D ECM, and further activates AKT/extracellular signal-regulated kinase 1/2 (ERK1/2) cascades, which directly regulates osteogenic differentiation of MSCs. Collectively, our findings implicate an intricate crosstalk between cell mechanics and metabolism, and provide new perspectives for strategies of osteoporosis.

Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a rate-limiting enzyme that plays critical roles in multiple physiological processes. The decompensation of PCK2 leads to various energy metabolic disorders. However, little is known regarding the effects of PCK2 on osteogenesis by human mesenchymal stem cells (hMSCs). Here, we report a novel function of PCK2 as a positive regulator of MSCs osteogenic differentiation. In addition to its well-known role in anabolism, we demonstrate that PCK2 regulates autophagy. PCK2 deficiency significantly suppressed autophagy, leading to the impairment of osteogenic capacity of MSCs. On the other hand, autophagy was promoted by PCK2 overexpression; this was accompanied by increased osteogenic differentiation of MSCs. Moreover, PCK2 regulated osteogenic differentiation of MSCs via AMP-activated protein kinase (AMPK)/unc-51 like autophagy activating kinase 1(ULK1)-dependent autophagy. Collectively, our present study unveiled a novel role for PCK2 in integrating autophagy and bone formation, providing a potential target for stem cell-based bone tissue engineering that may lead to improved therapies for metabolic bone diseases. Stem Cells 2019;37:1542–1555

The origin of life and native tissue development are dependent on the heterogeneity of pluripotent stem cells. Bone marrow mesenchymal stem cells (BMMSCs) are located in a complicated niche with variable matrix stiffnesses, resulting in divergent stem cell fates. However, how stiffness drives stem cell fate remains unknown. For this study, we performed whole-gene transcriptomics and precise untargeted metabolomics sequencing to elucidate the complex interaction network of stem cell transcriptional and metabolic signals in extracellular matrices (ECMs) with different stiffnesses, and we propose a potential mechanism involved in stem cell fate decision. In a stiff (39~45 kPa) ECM, biosynthesis of aminoacyl-tRNA was up-regulated, and increased osteogenesis was also observed. In a soft (7~10 kPa) ECM, biosynthesis of unsaturated fatty acids and deposition of glycosaminoglycans were increased, accompanied by enhanced adipogenic/chondrogenic differentiation of BMMSCs. In addition, a panel of genes responding to the stiffness of the ECM were validated in vitro, mapping out the key signaling network that regulates stem cells’ fate decisions. This finding of “stiffness-dependent manipulation of stem cell fate” provides a novel molecular biological basis for development of potential therapeutic targets within tissue engineering, from both a cellular metabolic and a biomechanical perspective.

The E3 ubiquitin ligase complex CDC20-activated anaphase-promoting complex/Cyclosome (APC/C ^CDC20 ) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co-activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well-known cell cycle-related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA-binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11-dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle-independent function of CDC20, establishes APC11 ^CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone-related diseases.

Organoids are widely considered to be ideal in vitro models that have been widely applied in many fields, including regenerative medicine, disease research and drug screening. It is distinguished from other three-dimensional (3D) in vitro culture model systems by self-organization and sustainability in long-term culture. The three core components of organoid culture are cells, exogenous factors and culture matrix. Due to the complexity of bone tissue, and heterogeneity of osteogenic stem/progenitor cells, it is challenging to construct organoids for modeling skeletal systems. Here, we examine current progress in the development of skeletal system organoid culture systems and provide cues for future research trajectory in the field by analyzing the current research status of skeletal stem cells, their microenvironmental factors and various potential organoid culture matrix candidates.