Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However, lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation remains poorly understood, and access to well-characterized placental cells for biomedical research is limited, largely depending on fetal tissues or cancer cell lines. Here, we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated syncytiotrophoblast (STB), followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological, biochemical, genomics, epigenomics, and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development, infertility, and pregnancy-associated diseases.
© 2024 The Author(s).

Human brain tissue models and organoids are vital for studying and modeling human neurological disease. However, the high cost of long-term cultured organoids inhibits their wide-ranging application. It is therefore urgent to develop methods for the cryopreservation of brain tissue and organoids. Here, we establish a method using methylcellulose, ethylene glycol, DMSO, and Y27632 (termed MEDY) for the cryopreservation of cortical organoids without disrupting the neural cytoarchitecture or functional activity. MEDY can be applied to multiple brain-region-specific organoids, including the dorsal/ventral forebrain, spinal cord, optic vesicle brain, and epilepsy patient-derived brain organoids. Additionally, MEDY enables the cryopreservation of human brain tissue samples, and pathological features are retained after thawing. Transcriptomic analysis shows that MEDY can protect synaptic function and inhibit the endoplasmic reticulum-mediated apoptosis pathway. MEDY will enable the large-scale and reliable storage of diverse neural organoids and living brain tissue and will facilitate wide-ranging research, medical applications, and drug screening.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Mitophagy regulates cancer stem cell (CSC) populations affecting tumorigenicity and malignancy in various cancer types. Here, we report that cisplatin treatment led to the activation of higher mitophagy through regulating CLU (clusterin) levels in oral CSCs. Moreover, both the gain-of-function and loss-of-function of CLU indicated its mitophagy-specific role in clearing damaged mitochondria. CLU also regulates mitochondrial fission by activating the Ser/Thr kinase AKT, which triggered phosphorylation of DNM1L/Drp1 at the serine 616 residue initiating mitochondrial fission. More importantly, we also demonstrated that CLU-mediated mitophagy positively regulates oral CSCs through mitophagic degradation of MSX2 (msh homeobox 2), preventing its nuclear translocation from suppressing SOX2 activity and subsequent inhibition of cancer stemness and self-renewal ability. However, CLU knockdown disturbed mitochondrial metabolism generating excessive mitochondrial superoxide, which improves the sensitivity to cisplatin in oral CSCs. Notably, our results showed that CLU-mediated cytoprotection relies on SOX2 expression. SOX2 inhibition through genetic (shSOX2) and pharmacological (KRX-0401) strategies reverses CLU-mediated cytoprotection, sensitizing oral CSCs toward cisplatin-mediated cell death.

The spinal cord possesses highly complex, finely organized cytoarchitecture guided by two dorsoventral morphogenic organizing centers. Thus, generation of human spinal cord tissue in vitro is challenging. Here, we demonstrated a novel method for generation of human dorsoventral spinal cord organoids using composite scaffolds. Specifically, the spinal cord ventralizing signaling Shh agonist (SAG) was loaded into a porous chitosan microsphere (PCSM), then thermosensitive Matrigel was coated on the surface to form composite microspheres with functional sustained-release SAG, termed as PCSM-Matrigel@SAG. Using PCSM-Matrigel@SAG as the core to induce 3D engineering of human spinal cord organoids from human pluripotent stem cells (ehSC-organoids), we found ehSC-organoids could form dorsoventral spinal cord-like cytoarchitecture with major domain-specific progenitors and neurons. Besides, these ehSC-organoids also showed functional calcium activity. In summary, these ehSC-organoids are of great significance for modeling spinal cord development, drug screening as 3D models for motor neuron diseases, and spinal cord injury repair.
© 2022 The Author(s).

The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that cross-talk between tumor and stromal cells within the tumor microenvironment results in activation of key biological pathways depending on their position in the tumor (edge vs. core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts from the tumor core, established from human lung adenocarcinomas. A multiomics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize cross-talk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential posttranslational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis in the stroma and indirectly modulates proinvasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma cross-talk and a potential avenue to improve the anticancer efficacy of CDK4 inhibitors.
A multiomics analysis of spatially distinct fibroblasts establishes the importance of the stromal O-glycoproteome in tumor-stroma interactions at the leading edge and provides potential strategies to improve cancer treatment. See related commentary by De Wever, p. 537.
©2021 American Association for Cancer Research.