Dysfunction of striatal medium spiny neurons (MSNs) is implicated in several neurological disorders, including Huntington’s disease (HD). Despite progress in characterizing MSN pathology in HD, mechanisms underlying MSN susceptibility remain unknown, driving the need for MSNs derived from human pluripotent stem cells (hPSCs), especially subtypes in research and therapy. Here, we established a scalable 3D-default culture system to produce striatal MSNs efficiently from hPSCs by activation of the endogenous sonic hedgehog (SHH) pathway. These cells expressed canonical markers of striatal progenitors and dopamine D1 (D1) and dopamine D2 (D2) MSNs and presented dynamic specification and transcriptional signatures that closely resemble endogenous MSNs at single-cell resolution, both in vitro and post-transplantation in HD mice with quinolinic acid (QA) lesions. Grafted human cells survived and matured into D1-/D2-like MSNs and projected axons to endogenous targets including globus pallidus externus, globus pallidus internus, and substantia nigra pars reticulata to reconstruct the basal ganglia pathways. Functionally, they displayed spontaneous synaptic currents, received regulation from host cortex and thalamus, and were modulated by dopamine to either enhance or reduce neuronal excitability, similar to the endogenous D1-/D2-MSNs, subsequently improving behavior in QA-lesioned HD mice. Our study presents a method for generating authentic MSNs, providing a reliable cell source for HD cell therapy, mechanistic studies, and drug screening.
Yuting Mei, Yuan Xu, Xinyue Zhang, Ban Feng, Yingying Zhou, Qian Cheng, Yuan Li, Xingsheng Peng, Mengnan Wu, Lianshun Xie, Lei Xiao, Wenhao Zhou, Yuejun Chen, Man Xiong
Stress-induced epithelial plasticity is central to lung regeneration, fibrosis, and malignancy, but how cellular stress leads to differentiation is incompletely understood. Here, we found a central role for IRE1α, a conserved mediator of the unfolded protein response (UPR), in stimulating the plasticity of alveolar type 2 (AT2) cells. In single-cell RNA-seq, IRE1α activity was associated with loss of AT2 identity and progression toward a damage-associated transitional state unique to fibrosis. AT2 plasticity required destructive regulated IRE1α-dependent decay (RIDD), which we demonstrated by deploying PAIR2, a kinase modulator that inhibits RIDD while preserving IRE1α’s adaptive XBP1 mRNA splicing activity. In vivo, selective inhibition of RIDD with PAIR2 reduced AT2 differentiation into profibrotic transitional cells and protected mice from bleomycin-induced pulmonary fibrosis. Mechanistically, we identified the Fgfr2 mRNA as a direct and regulated substrate for IRE1α’s RNase in primary AT2 cells and in a biochemically reconstituted cell-free system. Loss of Fgf signaling caused AT2 differentiation, while gain of signaling protected cells from IRE1α-induced differentiation. We propose that IRE1α downregulates Fgf signaling through RIDD, provoking loss of AT2 identity and differentiation towards a profibrotic phenotype. Thus, IRE1α’s RIDD activity emerges as a novel target for treatment of pulmonary fibrosis and potentially other diseases driven by aberrant epithelial cell plasticity.
Vincent C. Auyeung, Tavienne L. Steinberg, Alina Olivier, Luka Suzuki, Mary E. Moreno, Imran S. Khan, Michael S. Downey, Maike Thamsen, Lu Guo, Dustin J. Maly, Bradley J. Backes, Dean Sheppard, Feroz R. Papa
Chemotherapy resistance remains a formidable challenge to the treatment of high-grade serous ovarian cancer (HGSOC). The drug-tolerant cells may originate from a small population of inherently resistant cancer stem cells (CSCs) in primary tumors. In contrast, sufficient evidence suggests that drug tolerance can also be transiently acquired by nonstem cancer cells. Regardless of the route, key regulators of this plastic process are poorly understood. Here, we utilized multiomics, tumor microarrays, and epigenetic modulation to demonstrate that SOX9 is a key chemo-induced driver of chemoresistance in HGSOC. Epigenetic upregulation of SOX9 was sufficient to induce chemoresistance in multiple HGSOC lines. Moreover, this upregulation induced the formation of a stem-like subpopulation and significant chemoresistance in vivo. Mechanistically, SOX9 increased transcriptional divergence, reprogramming the transcriptional state of naive cells into a stem-like state. Supporting this, we identified a rare cluster of SOX9-expressing cells in primary tumors that were highly enriched for CSCs and chemoresistance-associated stress gene modules. Notably, single-cell analysis showed that chemo treatment results in rapid population-level induction of SOX9 that enriches for a stem-like transcriptional state. Altogether, these findings implicate SOX9 as a critical regulator of early steps of transcriptional reprogramming that lead to chemoresistance through a CSC-like state in HGSOC.
Alexander J. Duval, Fidan Seker-Polat, Magdalena Rogozinska, Meric Kinali, Ann E. Walts, Ozlem Neyisci, Yaqi Zhang, Zhonglin Li, Edward J. Tanner III, Allison E. Grubbs, Sandra Orsulic, Daniela Matei, Mazhar Adli
The balance of hematopoietic stem cell (HSC) self-renewal versus differentiation is essential to ensure long-term repopulation capacity while allowing response to events that require increased hematopoietic output. Proliferation and differentiation of HSCs and their progeny is controlled by the JAK/STAT pathway downstream of cytokine signaling. E3 ubiquitin ligases, like Cullin 5 (Cul5), can regulate JAK/STAT signaling by degrading signaling intermediates. Here we report that mice lacking Cul5 in hematopoietic cells (Cul5Vav-Cre) have increased numbers of HSPCs, splenomegaly, and extramedullary hematopoiesis. Differentiation in Cul5Vav-Cre mice is myeloid- and megakaryocyte-biased, resulting in leukocytosis, anemia and thrombocytosis. Cul5Vav-Cre mice increased HSC proliferation and circulation, associated with a decrease in CXCR4 surface expression. In bone marrow cells, we identified LRRC41 co-immunoprecipitated with CUL5, and vice versa, supporting that CRL5 forms a complex with LRRC41. We identified an accumulation of LRRC41 and STAT5 in Cul5Vav-Cre HSCs during IL-3 stimulation, supporting their regulation by Cul5. Whole cell proteome (WCP) analysis of HSPCs from Cul5Vav-Cre bone marrow identified upregulation of many STAT5 target genes and associated pathways. Finally, JAK1/2 inhibition with ruxolitinib normalized hematopoiesis in Cul5Vav-Cre mice. These studies demonstrate the function of Cul5 in HSC function, stem cell fate decisions, and regulation of IL-3 signaling.
Siera A. Tomishima, Dale D. Kim, Nadia Porter, Ipsita Guha, Asif A. Dar, Yohaniz Ortega-Burgos, Jennifer Roof, Hossein Fazelinia, Lynn A. Spruce, Christopher S. Thom, Robert L. Bowman, Paula M. Oliver
BACKGROUND. Telomere biology disorders (TBDs) exhibit incomplete penetrance and variable expressivity, even among individuals harboring the same pathogenic variant. We assessed whether common genetic variants associated with telomere length combine with large-effect variants to impact penetrance and expressivity in TBDs. METHODS. We constructed polygenic scores (PGS) for telomere length in the UK Biobank to quantify common variant burden, and assessed the PGS distribution across patient cohorts and biobanks to determine whether individuals with severe TBD presentations have increased polygenic burden causing short telomeres. We also characterized rare TBD variant carriers in the UK Biobank. RESULTS. Individuals with TBDs in cohorts enriched for severe pediatric presentations have polygenic scores predictive of short telomeres. In the UK Biobank, we identify carriers of pathogenic TBD variants who are enriched for adult-onset manifestations of TBDs. Unlike individuals in disease cohorts, the PGS of adult carriers do not show a common variant burden for shorter telomeres, consistent with the absence of childhood-onset disease. Notably, TBD variant carriers are enriched for idiopathic pulmonary fibrosis diagnoses, and telomere length PGS stratifies pulmonary fibrosis risk. Finally, common variants affecting telomere length were enriched in enhancers regulating known TBD genes. CONCLUSION. Common genetic variants combine with large-effect causal variants to impact clinical manifestations in rare TBDs. These findings offer a framework for understanding phenotypic variability in other presumed monogenic disorders. FUNDING. This work was supported by National Institutes of Health grants R01DK103794, R01HL146500, R01CA265726, R01CA292941, and the Howard Hughes Medical Institute.
Michael Poeschla, Uma P. Arora, Amanda Walne, Lisa J. McReynolds, Marena R. Niewisch, Neelam Giri, Logan P. Zeigler, Alexander Gusev, Mitchell J. Machiela, Hemanth Tummala, Sharon A. Savage, Vijay G. Sankaran
Mechanisms by which mucosal regeneration is abrogated in inflammatory bowel disease (IBD) are still under investigation, and a role for an intestinal stem cell (ISC) defect is now emerging. Herein, we report an abnormal ISC death that occurs in Crohn’s disease, which exacerbates colitis, limits ISC-dependent mucosal repair, and is controlled through the death factor Transmembrane protein 219 (TMEM219). Large alterations in TMEM219 expression were observed in patients with Crohn’s disease, particularly in those with active disease and/or those who were nonresponders to conventional therapy, confirming that TMEM219 signaling is abnormally activated and leads to failure of the mucosal regenerative response. Mechanistic studies revealed a proapoptotic TMEM219-mediated molecular signature in Crohn’s disease, which associates with Caspase-8 activation and ISC death. Pharmacological blockade of the IGFBP3/TMEM219 binding/signal with the recombinant protein ecto-TMEM219 restored the self-renewal abilities of miniguts generated from patients with Crohn’s disease in vitro and ameliorated DSS-induced and T cell-mediated colitis in vivo, ultimately leading to mucosal healing. Genetic tissue-specific deletion of TMEM219 in ISCs in newly generated TMEM219fl/flLGR5cre mice revived their mucosal regenerative abilities both in vitro and in vivo. Our findings demonstrate that a TMEM219-dependent ISC death exacerbates colitis and that TMEM219 blockade reestablishes intestinal self-renewal properties in IBD.
Francesca D’Addio, Giovanni Amabile, Emma Assi, Anna Maestroni, Adriana Petrazzuolo, Cristian Loretelli, Ahmed Abdelasalam, Moufida Ben Nasr, Ida Pastore, Maria Elena Lunati, Vera Usuelli, Monica Zocchi, Andy Joe Seelam, Domenico Corradi, Stefano La Rosa, Virna Marin, Monique Zangarini, Marta Nardini, Stefano Porzio, Filippo Canducci, Claudia Nardini, Basset El Essawy, Manuela Nebuloni, Jun Yang, Massimo Venturini, Giovanni Maconi, Franco Folli, Silvio Danese, Gianvincenzo Zuccotti, Gianluca M. Sampietro, Sandro Ardizzone, Paolo Fiorina
The nucleolus is a membraneless organelle and an excellent stress sensor. Any changes in its architecture or composition lead to nucleolar stress, resulting in cell cycle arrest and interruption of ribosomal activity, critical factors in aging and cancer. In this study, we identified and described the pivotal role of the RNA-binding protein (RBP) HNRNPK in ribosome and nucleolar dynamics. We developed an in vitro model of endogenous HNRNPK overexpression and an in vivo mouse model of ubiquitous HNRNPK overexpression. These models showed disruptions in translation and caused alterations in the nucleolar structure, resulting in p53-dependent nucleolar stress, cell cycle arrest, senescence, and bone marrow failure phenotype, similar to what is observed in patients with ribosomopathies. Together, our findings identify HNRNPK as a master regulator of ribosome biogenesis (RiBi) and nucleolar homeostasis through p53, providing a new perspective on the orchestration of nucleolar integrity, ribosome function and cellular senescence.
Pedro Aguilar-Garrido, María Velasco-Estévez, Miguel Ángel Navarro-Aguadero, Alvaro Otero-Sobrino, Marta Ibañez-Navarro, Miguel Ángel Marugal, María Hernández-Sánchez, Prerna Malaney, Ashley Rodriguez, Oscar Benitez, Xiaorui Zhang, Marisa J.L. Aitken, Alejandra Ortiz-Ruiz, Diego Megias, Manuel Pérez-Martínez, Gadea Mata, Jesús Gomez, Miguel Lafarga, Orlando Dominguez, Osvaldo Graña-Castro, Eduardo Caleiras, Pilar Ximenez-Embun, Marta Isasa, Paloma J. de Andrés, Sandra Rodriguez-Perales, Raul Torres-Ruiz, Enrique Revilla, Rosa María García-Martín, Daniel Azorín, Josune Zubicaray, Julian Sevilla, Oleksandra Sirozh, Vanesa Lafarga, Joaquín Martinez-Lopez, Sean M. Post, Miguel Gallardo
Umbilical cord blood (UCB) showcases substantial roles in hematopoietic stem cells (HSCs) transplantation and regenerative medicine. UCB is usually cryopreserved for years before use. Whether and how cryopreservation affects its function remain unclear. We constructed single-cell transcriptomic profile of CD34+ hematopoietic stem and progenitor cells (HSPCs) and mononuclear cells (MNCs) from fresh and cryopreserved UCB stored for 1-, 5-, 10-, and 19- years. Compared to fresh UCB, cryopreserved HSCs and multipotent progenitors (MPPs) exhibited more active cell cycle and lower HSC/MPP signature gene expressions. Hematopoietic reconstitution of cryopreserved HSPCs gradually decreased during the first 5 years but stabilized thereafter, aligning with the negative correlation between clinical neutrophil engraftment and cryopreservation duration of UCB. Cryopreserved HSPCs also showed reduced megakaryocyte generation. In contrast, cryopreserved natural killer (NK) cells and T cells maintained cytokine production and cytotoxic ability comparable to fresh cells. Mechanistically, cryopreserved HSPCs exhibited elevated reactive oxygen species, reduced ATP synthesis, and abnormal mitochondrial distribution, which collectively led to attenuated hematopoietic reconstitution. These effects could be ameliorated by sulforaphane. Together, we elucidated the negative impact of cryopreservation on UCB HSPCs and provided sulforaphane as a mitigation strategy, broadening the temporal window and scope for clinical applications of cryopreserved UCB.
Yaojin Huang, Xiaowei Xie, Mengyao Liu, Yawen Zhang, Junye Yang, Wenling Yang, Yu Hu, Saibing Qi, Yahui Feng, Guojun Liu, Shihong Lu, Xuemei Peng, Jinhui Ye, Shihui Ma, Jiali Sun, Lu Wang, Linping Hu, Lin Wang, Xiaofan Zhu, Hui Cheng, Zimin Sun, Junren Chen, Fang Dong, Yingchi Zhang, Tao Cheng
Newly produced platelets acquire a low activation state but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found healthy megakaryocytes transfer mitochondria to MSCs mediated by Connexin 43 (Cx43) gap junctions on MSCs, which leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human patient samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.
Chengjie Gao, Yitian Dai, Paul A. Spezza, Paul Boasiako, Alice Tang, Giselle Rasquinha, Hui zhong, Bojing Shao, Yunfeng Liu, Patricia A. Shi, Cheryl A. Lobo, Xiuli An, Anqi Guo, William B. Mitchell, Deepa Manwani, Karina Yazdanbakhsh, Avital Mendelson
The bone marrow (BM) niche is critical in regulating hematopoiesis, and sexual dimorphism and its underlying mechanism in BM niche and its impact on hematopoiesis are not well understood. We show that male mice exhibited a higher abundance of leptin-receptor-expressing mesenchymal stromal cells (LepR-MSCs) compared to female mice. Sex-mismatched co-culture and BM transplantation showed that the male BM niche provided superior support for in vitro colony formation and in vivo hematopoietic engraftment. The co-transplantation of male stromal cells significantly enhanced engraftment in female recipients. Single-cell RNA sequencing revealed that the lower expression of the X-linked lysine H3K4 demethylase, Kdm5c, in male MSCs led to the increased expression of Cxcl12. In MSC-specific Kdm5c knockout mouse model, the reduction of KDM5C in female MSCs enhanced MSC quantity and function, ultimately improving engraftment to the male level. Kdm5c thus plays a role in driving sexual dimorphism in the BM niche and hematopoietic regeneration. Our study unveils a sex-dependent mechanism governing BM niche regulation and its impact on hematopoietic engraftment. The finding offers potential implications for enhancing BM transplantation efficacy in clinical settings by harnessing the resource of male MSCs or targeting Kdm5c.
Xiaojing Cui, Liming Hou, Bowen Yan, Jinpeng Liu, Cuiping Zhang, Pinpin Sui, Sheng Tong, Larry Luchsinger, Avital Mendelson, Daohong Zhou, Feng-chun Yang, Hui zhong, Ying Liang