Dae-Sung Kim

Ventral midbrain dopaminergic (vmDA) neurons derived from human pluripotent stem cells provide a powerful platform for modeling Parkinson's disease (PD) and developing therapeutic strategies. Although robust protocols exist for generating vmDA progenitors, maturation into functionally competent neurons typically requires prolonged culture periods and occurs with substantial heterogeneity in maturity, representing major bottlenecks. Building on established protocols for vmDA progenitor differentiation, we introduce a sequential strategy that specifically targets the subsequent phases of neuronal conversion and postmitotic maturation. Transient overexpression of the proneural transcription factor achaete-scute family bHLH transcription factor 1 (ASCL1) was applied to human embryonic stem cell-derived vmDA progenitors to induce neuronal commitment, followed by accelerated postmitotic maturation using the small-molecule cocktail GENtoniK. This approach was evaluated in CRISPR/Cas9-engineered human induced pluripotent stem cell (hiPSC) lines lacking either PINK1 or PRKN and their isogenic control hiPSCs. Transient ASCL1 overexpression promoted rapid cell cycle exit and neuronal conversion of vmDA progenitors while preserving ventral midbrain identity and dopaminergic fate. Subsequent GENtoniK treatment enhanced postmitotic dopaminergic maturation and increased neurite complexity, synaptic organization, and dopaminergic neurotransmission without altering lineage specification. Consequently, within two weeks, our protocol yielded high-purity vmDA neurons with enhanced functional maturation from vmDA progenitors. Importantly, PINK1- and PRKN-deficient neurons generated from CRISPR/Cas9-engineered hiPSC lines using this strategy recapitulated key PD-associated mitochondrial phenotypes, including impaired mitophagy, mitochondrial dysfunction, and elevated oxidative stress. Taken together, these findings address the central limitations of current dopaminergic differentiation paradigms by enabling the rapid and uniform acquisition of advanced functional maturity, thereby providing a robust platform for disease modeling and translational research in PD.

This study explored the role of telomere attrition in astrocytic senescence by pharmacologically inhibiting telomerase activity in human induced pluripotent stem cell‐derived astrocytes. Treatment with the telomerase inhibitor BIBR1532 (BIBR) during differentiation induced hallmark features of senescence, including nuclear lamina abnormalities, enhanced senescence‐associated β‐galactosidase activity, increased replication arrest and DNA damage, altered reactive oxygen species homeostasis in mitochondria, accompanied by significant shortening of relative telomere length. Despite these senescence related characteristics, BIBR‐treated astrocytes exhibited limited changes in the expression of senescence‐associated secretory phenotype‐related genes. Moreover, their key functional properties, such as glutamate uptake, synaptic vesicle clearance, mitochondrial membrane potential and morphology remain comparable to those of control astrocytes. These findings suggest that the presence of classical senescence markers does not necessarily lead to functional impairment and that BIBR‐induced senescence in astrocytes may represent an early or transitional phase, where classical senescence markers emerge without substantial functional decline. Our results reinforce the notion that while telomere attrition is a major cellular senescence driver, its onset may not be attributed to a single stressor but rather to a complex interplay of cellular stress pathways. This study provides valuable insights into the mechanisms underlying astrocytic senescence and underscores the need for further research on the molecular basis of its occurrence and functional implications.

Metabolic alterations have been observed in the brains of patients with bipolar disorder (BD), a neuropsychiatric disorder characterized by alternating episodes of mania and depression. However, the specific contributions of glial cells to these metabolic changes remain largely unknown. Here, we investigate the metabolic characteristics of induced astrocytes (iAstrocytes) derived from induced pluripotent stem cells of BD patients—classified by lithium responsiveness—and healthy controls. Transcriptomic analyses revealed dysregulated expression of genes associated with metabolic diseases in BD iAstrocytes. Compared to control iAstrocytes, BD iAstrocytes showed decreased mitochondrial respiration, increased glycolysis, and elevated lactate secretion, indicating impaired mitochondrial function. These defects were further supported by downregulation of oxidative phosphorylation complex proteins and decreased reactive oxygen species levels. Notably, BD iAstrocytes showed substantial lipid droplet (LD) accumulation, potentially as a consequence of disrupted metabolic homeostasis. Lithium treatment reduced LD levels in lithium-responsive (Li-R) iAstrocytes but failed to restore mitochondrial respiration or normalize lactate secretion. In co-culture with human neurons, lithium-nonresponsive (Li-NR) iAstrocytes exhibited enhanced uptake of neuron-derived lipids and selectively increased neuronal excitability. Metabolomic profiling revealed distinct metabolite signatures between Li-R and Li-NR iAstrocytes, suggesting lithium responsiveness as a key axis of metabolic heterogeneity. Together, our findings identify astrocyte-specific metabolic dysfunction as a hallmark of BD and reveal divergent roles of Li-R and Li-NR iAstrocytes in modulating neuronal function. LD accumulation in Li-NR iAstrocytes may serve as a functional readout for drug screening to identify alternative treatments for patients unresponsive to lithium.

To elucidate the complex interplay of undifferentiated cancer cells in malignancy, we focus on the crucial mechanisms that maintain the undifferentiated state of cancer stem-like cells, which drive tumor growth and therapy resistance. Here, we identify a protein called dehydrogenase/reductase 13 (DHRS13) that is abundant in undifferentiated glioblastoma cells. DHRS13 is primarily located in the mitochondria and functions as a retinaldehyde reductase, converting all-trans-retinaldehyde to all-trans-retinol with high affinity for NADPH. Mechanistically, DHRS13 prevents glioma stem-like cells from differentiating by blocking retinoic acid signaling, thereby maintaining their undifferentiated state. Remarkably, the depletion of DHRS13 results in mitochondrial reactive oxygen species-driven mitophagy and cell death. Consequently, loss of DHRS13 leads to a significant decrease in tumor initiation and progression. These findings hold promise for the development of strategies that target undifferentiated cancer cells, potentially leading to improved treatment outcomes.

Hemophilia A (HA) is caused by mutations in coagulation factor VIII ( FVIII ). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)- FVIII ( FE-FVIII ), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII -inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII -containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.

Astrocytes play a crucial role in maintaining brain homeostasis by regulating synaptic activity, providing metabolic support to neurons, and modulating immune responses in the central nervous system (CNS). During aging, astrocytes undergo senescence with various changes that affect their function and frequently lead to neurodegeneration. This study presents the first evidence of senescent astrocytes derived from human pluripotent stem cells (hPSCs). These senescent hPSC-derived astrocytes exhibited altered cellular and nuclear morphologies, along with increased expression of senescence-associated markers. Additionally, nuclear localization of NFκB, telomere shortening, and frequent signs of DNA damage were observed in these cells. Furthermore, senescent astrocytes showed defects in various critical functions necessary for maintaining a healthy CNS environment, including a reduced ability to support neuronal survival and clear neurotransmitters, synaptic debris, and toxic protein aggregates. Altered structural dynamics and reduced mitochondrial function were also observed in senescent astrocytes. Notably, treating hPSC-derived senescent astrocytes with chemicals targeting reactive oxygen species or an enzyme that regulates mitochondrial function can reverse senescence phenotypes. Thus, this study offers a valuable cellular model that can be utilized to investigate the mechanisms of brain aging and may present new avenues for discovering innovative therapeutic approaches for neurodegenerative diseases.

Molecular and functional diversity among region-specific astrocytes is of great interest in basic neuroscience and the study of neurological diseases. In this study, we present the generation and characterization of astrocytes from human embryonic stem cells with the characteristics of the ventral midbrain (VM). Fine modulation of WNT and SHH signaling during neural differentiation induced neural precursor cells (NPCs) with high expression of EN1 and NKX6.1, but less expression of FOXA2. Overexpression of nuclear factor IB in NPCs induced astrocytes, thereby maintaining the expression of region-specific genes acquired in the NPC stage. When cocultured with dopaminergic (DA) precursors or DA neurons, astrocytes with VM characteristics (VM-iASTs) promoted the differentiation and survival of DA neurons better than those that were not regionally specified. Transcriptomic analysis showed that VM-iASTs were more closely related to human primary midbrain astrocytes than to cortical astrocytes, and revealed the upregulation of WNT1 and WNT5A, which supports their VM identity and explains their superior activity in DA neurons. Taken together, we hope that VM-iASTs can serve to improve ongoing DA precursor transplantation for Parkinson’s disease, and that their transcriptomic data provide a valuable resource for investigating regional diversity in human astrocyte populations. Graphical Abstract