The mitochondrial genome (mtDNA) is an important source of inherited extranuclear variation. Clonal increases in mtDNA mutation heteroplasmy have been implicated in aging and disease, although the impact of this shift on cell function is challenging to assess. Reprogramming to pluripotency affects mtDNA mutation heteroplasmy. We reprogrammed three human fibroblast lines with known heteroplasmy for deleterious mtDNA point or deletion mutations. Quantification of mutation heteroplasmy in the resulting 76 induced pluripotent stem cell (iPSC) clones yielded a bimodal distribution, creating three sets of clones with high levels or absent mutation heteroplasmy with matched nuclear genomes. iPSC clones with elevated deletion mutation heteroplasmy show altered growth dynamics, which persist in iPSC-derived progenitor cells. We identify transcriptomic and metabolic shifts consistent with increased investment in neutral lipid synthesis as well as increased epigenetic age in high mtDNA deletion mutation iPSC, consistent with changes occurring in cellular aging. Together, these data demonstrate that high mtDNA mutation heteroplasmy induces changes occurring in cellular aging.
© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Cellular plasticity plays critical roles in tissue regeneration, tumour progression and therapeutic resistance. However, the mechanism underlying this cell state transition remains elusive. Here, we show that the transcription factor SOX2 induces fetal reprogramming and reversible dormancy in colorectal cancer (CRC). SOX2 expression correlates with fetal reprogramming and poor prognosis in human primary and metastatic colorectal adenocarcinomas. In mouse CRC models, rare slow-cycling SOX2 + SCA1 + cells are detected in early Apc -deleted mouse tumours that undergo slow clonal expansion over time. In contrast, the SOX2 + clones were found proliferative in advanced Apc -/- ;Kras G12D/+ ;p53 -/- ;Tgfbr2 -/- (AKPT) tumours, accompanied by dynamic cell state reprogramming from LGR5 + to LGR5 - SCA1 + cells. Using transgenic mouse models, we demonstrate that ectopic expression of SOX2 inhibits intestinal lineage differentiation and induces fetal reprogramming. SOX2+ cells adopt dynamic cell cycle states depending on its expression level. High SOX2 expression results in hyperproliferation, whereas low SOX2 levels induce senescence-mediated dormancy. We find that loss of p53 can reverse SOX2-induced senescence, in line with the dormant cell state exit of the SOX2+ cells observed in advanced tumours. Finally, SOX2 expression is induced by 5-FU treatment in CRC. SOX2-expressing organoids exhibit increased tolerance to chemotherapy treatment, whilst deletion of SOX2 in AKPT tumour organoids sensitises drug responses. We propose that SOX2-induced plasticity and reversible dormancy promotes tumour progression and drug tolerance in CRC.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system that mediate nerve conduction. Loss of oligodendrocytes results in demyelination, triggering neurological deficits. Developing a better understanding of the cell signaling pathways influencing OL development may aid in the development of therapeutic strategies. The primary focus of this study was to investigate and elucidate the cell signaling pathways implicated in the developmental maturation of oligodendrocytes using human fetal neural stem cells (hFNSCs)-derived primary OL and MO3.13 cell line. Successful differentiation into OL was established by examining morphological changes, increased expression of mature OL markers MBP, MOG and decreased expression of pre-OL markers CSPG4 and O4. Analyzing transcriptional datasets (using RNA sequencing) in pre-OL and mature OL derived from hFNSCs revealed the novel and critical involvement of the JAK-STAT cell signaling pathway in terminal OL maturation. The finding was validated in MO3.13 cell line whose differentiation was accompanied by upregulation of IL-6 and the transcription factor STAT3. Increased phosphorylated STAT3 (pY705) levels were demonstrated by western blotting in hFNSCs-derived primary OL as well as terminal maturation in MO3.13 cells, thus validating the involvement of the JAK-STAT pathway in OL maturation. Pharmacological suppression of STAT3 phosphorylation (confirmed by western blotting) was able to prevent the increase of MBP-positive cells as demonstrated by flow cytometry. These novel findings highlight the involvement of the JAK-STAT pathway in OL maturation and raise the possibility of using this as a therapeutic strategy in demyelinating diseases.
© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

ABSTRACT The speed of embryonic development varies considerably between mammalian species, yet the underlying molecular mechanisms remain poorly understood. To investigate the basis for species-specific developmental tempo, we performed a comprehensive comparative analysis of protein dynamics in mouse and human neural progenitors (NPs). Through a combination of targeted protein labelling, quantitative mass spectrometry, and protein depletion with self-labeling tags, we demonstrate that protein degradation is a key driver of tempo differences between mouse and human NPs. We observe a systematic 1.5-fold increase in protein half-lives in human NPs compared to mouse, independent of cellular compartment or protein function. This difference persists in post-mitotic neurons, indicating active degradation as the primary mechanism. Proteasomal activity is also ∼1.5-fold higher in mouse NPs, consistent with upregulation of proteasome-associated proteins. Importantly, increasing the rate of proteolytic degradation of a key transcriptional repressor in neural progenitors accelerates the expression of its target gene. Despite differences in degradation rates, protein synthesis rates are similar between species, resulting in higher protein content in human NPs. Our findings highlight the central role of protein degradation in controlling developmental tempo and provide insight into the molecular basis of evolutionary changes in developmental timing across species.

Although current stem cell therapies exhibit promising potential, the extended process of employing autologous cells and the necessity for donor-host matching to avert the rejection of transplanted cells significantly limit the widespread applicability of these treatments. It would be highly advantageous to generate a pluripotent universal donor stem cell line that is immune-evasive and, therefore, not restricted by the individual's immune system, enabling unlimited application within cell replacement therapies. Before such immune-evasive stem cells can be moved forward to clinical trials, in vivo testing via transplantation experiments in immune-competent animals would be a favorable approach preceding preclinical testing. By using human stem cells in immune competent animals, results will be more translatable to a clinical setting, as no parts of the immune system have been altered, although in a xenogeneic setting. In this way, immune evasiveness, cell survival, and unwanted proliferative effects can be assessed before clinical trials in humans. The current study presents the generation and characterization of three human embryonic stem cell lines (hESCs) for xenogeneic transplantation in immune-competent mice. The major histocompatibility complexes I- and II-encoding genes, B2M and CIITA, have been deleted from the hESCs using CRISPR-Cas9-targeted gene replacement strategies and knockout. B2M was knocked out by the insertion of murine CD47. Human-secreted embryonic alkaline phosphatase (hSEAP) was inserted in a safe harbor site to track cells in vivo. The edited hESCs maintained their pluripotency, karyotypic normality, and stable expression of murine CD47 and hSEAP in vitro. In vivo transplantation of hESCs into immune-competent BALB/c mice was successfully monitored by measuring hSEAP in blood samples. Nevertheless, transplantation of immune-evasive hESCs resulted in complete rejection within 11 days, with clear immune infiltration of T-cells on day 8. Our results reveal that knockout of B2M and CIITA together with species-specific expression of CD47 are insufficient to prevent rejection in an immune-competent and xenogeneic context.
Copyright © 2024 Frederiksen, Glantz, Vøls, Skov, Tveden-Nyborg, Freude and Doehn.