Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

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Abstract

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1–expressing (NKX2-1⁺) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1^GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1⁺ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47^hiCD26^lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

Authors

Finn Hawkins, Philipp Kramer, Anjali Jacob, Ian Driver, Dylan C. Thomas, Katherine B. McCauley, Nicholas Skvir, Ana M. Crane, Anita A. Kurmann, Anthony N. Hollenberg, Sinead Nguyen, Brandon G. Wong, Ahmad S. Khalil, Sarah X.L. Huang, Susan Guttentag, Jason R. Rock, John M. Shannon, Brian R. Davis, Darrell N. Kotton

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction

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Abstract

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2⁺) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2⁺ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2⁺ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2⁺ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Authors

Christa F. Gaskill, Erica J. Carrier, Jonathan A. Kropski, Nathaniel C. Bloodworth, Swapna Menon, Robert F. Foronjy, M. Mark Taketo, Charles C. Hong, Eric D. Austin, James D. West, Anna L. Means, James E. Loyd, W. David Merryman, Anna R. Hemnes, Stijn De Langhe, Timothy S. Blackwell, Dwight J. Klemm, Susan M. Majka

Disruption of spatiotemporal hypoxic signaling causes congenital heart disease in mice

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Abstract

Congenital heart disease (CHD) represents the most prevalent inborn anomaly. Only a minority of CHD cases are attributed to genetic causes, suggesting a major role of environmental factors. Nonphysiological hypoxia during early pregnancy induces CHD, but the underlying reasons are unknown. Here, we have demonstrated that cells in the mouse heart tube are hypoxic, while cardiac progenitor cells (CPCs) expressing islet 1 (ISL1) in the secondary heart field (SHF) are normoxic. In ISL1⁺ CPCs, induction of hypoxic responses caused CHD by repressing Isl1 and activating NK2 homeobox 5 (Nkx2.5), resulting in decreased cell proliferation and enhanced cardiomyocyte specification. We found that HIF1α formed a complex with the Notch effector hes family bHLH transcription factor 1 (HES1) and the protein deacetylase sirtuin 1 (SIRT1) at the Isl1 gene. This complex repressed Isl1 in the hypoxic heart tube or following induction of ectopic hypoxic responses. Subsequently, reduced Isl1 expression abrogated ISL1-dependent recruitment of histone deacetylases HDAC1/5, inhibiting Nkx2.5 expression. Inactivation of Sirt1 in ISL1⁺ CPCs blocked Isl1 suppression via the HIF1α/HES1/SIRT1 complex and prevented CHDs induced by pathological hypoxia. Our results indicate that spatial differences in oxygenation of the developing heart serve as signals to control CPC expansion and cardiac morphogenesis. We propose that physiological hypoxia coordinates homeostasis of CPCs, providing mechanistic explanations for some nongenetic causes of CHD.

Authors

Xuejun Yuan, Hui Qi, Xiang Li, Fan Wu, Jian Fang, Eva Bober, Gergana Dobreva, Yonggang Zhou, Thomas Braun

Amino acid–insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells

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Abstract

The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches.

Authors

Demetrios Kalaitzidis, Dongjun Lee, Alejo Efeyan, Youmna Kfoury, Naema Nayyar, David B. Sykes, Francois E. Mercier, Ani Papazian, Ninib Baryawno, Gabriel D. Victora, Donna Neuberg, David M. Sabatini, David T. Scadden

FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

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Abstract

A hallmark of aged mesenchymal stem/progenitor cells (MSCs) in bone marrow is the pivot of differentiation potency from osteoblast to adipocyte coupled with a decrease in self-renewal capacity. However, how these cellular events are orchestrated in the aging progress is not fully understood. In this study, we have used molecular and genetic approaches to investigate the role of forkhead box P1 (FOXP1) in transcriptional control of MSC senescence. In bone marrow MSCs, FOXP1 expression levels declined with age in an inverse manner with those of the senescence marker p16^INK4A. Conditional depletion of Foxp1 in bone marrow MSCs led to premature aging characteristics, including increased bone marrow adiposity, decreased bone mass, and impaired MSC self-renewal capacity in mice. At the molecular level, FOXP1 regulated cell-fate choice of MSCs through interactions with the CEBPβ/δ complex and recombination signal binding protein for immunoglobulin κ J region (RBPjκ), key modulators of adipogenesis and osteogenesis, respectively. Loss of p16^INK4A in Foxp1-deficient MSCs partially rescued the defects in replication capacity and bone mass accrual. Promoter occupancy analyses revealed that FOXP1 directly represses transcription of p16^INK4A. These results indicate that FOXP1 attenuates MSC senescence by orchestrating their cell-fate switch while maintaining their replicative capacity in a dose- and age-dependent manner.

Authors

Hanjun Li, Pei Liu, Shuqin Xu, Yinghua Li, Joseph D. Dekker, Baojie Li, Ying Fan, Zhenlin Zhang, Yang Hong, Gong Yang, Tingting Tang, Yongxin Ren, Haley O. Tucker, Zhengju Yao, Xizhi Guo

Versatile humanized niche model enables study of normal and malignant human hematopoiesis

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Abstract

The BM niche comprises a tightly controlled microenvironment formed by specific tissue and cells that regulates the behavior of hematopoietic stem cells (HSCs). Here, we have provided a 3D model that is tunable in different BM niche components and useful, both in vitro and in vivo, for studying the maintenance of normal and malignant hematopoiesis. Using scaffolds, we tested the capacity of different stromal cell types to support human HSCs. Scaffolds coated with human mesenchymal stromal cells (hMSCs) proved to be superior in terms of HSC engraftment and long-term maintenance when implanted in vivo. Moreover, we found that hMSC-coated scaffolds can be modulated to form humanized bone tissue, which was also able to support human HSC engraftment. Importantly, hMSC-coated humanized scaffolds were able to support the growth of leukemia patient cells in vivo, including the growth of samples that would not engraft the BM of immunodeficient mice. These results demonstrate that an s.c. implantation approach in a 3D carrier scaffold seeded with stromal cells is an effective in vivo niche model for studying human hematopoiesis. The various niche components of this model can be changed depending on the context to improve the engraftment of nonengrafting acute myeloid leukemia (AML) samples.

Authors

Ander Abarrategi, Katie Foster, Ashley Hamilton, Syed A. Mian, Diana Passaro, John Gribben, Ghulam Mufti, Dominique Bonnet

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

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Abstract

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.

Authors

Hao Gu, Chiqi Chen, Xiaoxin Hao, Conghui Wang, Xiaocui Zhang, Zhen Li, Hongfang Shao, Hongxiang Zeng, Zhuo Yu, Li Xie, Fangzhen Xia, Feifei Zhang, Xiaoye Liu, Yaping Zhang, Haishan Jiang, Jun Zhu, Jiangbo Wan, Chun Wang, Wei Weng, Jingjing Xie, Minfang Tao, Cheng Cheng Zhang, Junling Liu, Guo-Qiang Chen, Junke Zheng

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

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Abstract

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.

Authors

Bastien Gerby, Diogo F.T. Veiga, Jana Krosl, Sami Nourreddine, Julianne Ouellette, André Haman, Geneviève Lavoie, Iman Fares, Mathieu Tremblay, Véronique Litalien, Elizabeth Ottoni, Milena Kosic, Dominique Geoffrion, Joël Ryan, Paul S. Maddox, Jalila Chagraoui, Anne Marinier, Josée Hébert, Guy Sauvageau, Benjamin H. Kwok, Philippe P. Roux, Trang Hoang

Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice

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Abstract

Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5⁺ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G₁/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis–dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection.

Authors

Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu

COUP-TFII regulates satellite cell function and muscular dystrophy

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Abstract

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disease caused by mutations in the dystrophin gene. Although dystrophin deficiency in myofiber triggers the disease’s pathological changes, the degree of satellite cell (SC) dysfunction defines disease progression. Here, we have identified chicken ovalbumin upstream promoter–transcription factor II (COUP-TFII) hyperactivity as a contributing factor underlying muscular dystrophy in a dystrophin-deficient murine model of DMD. Ectopic expression of COUP-TFII in murine SCs led to Duchenne-like dystrophy in the muscles of control animals and exacerbated degenerative myopathies in dystrophin-deficient mice. COUP-TFII–overexpressing mice exhibited regenerative failure that was attributed to deficient SC proliferation and myoblast fusion. Mechanistically, we determined that COUP-TFII coordinated a regenerative program through combined regulation of multiple promyogenic factors. Furthermore, inhibition of COUP-TFII preserved SC function and counteracted the muscle weakness associated with Duchenne-like dystrophy in the murine model, suggesting that targeting COUP-TFII is a potential treatment for DMD. Together, our findings reveal a regulatory role of COUP-TFII in the development of muscular dystrophy and open up a potential therapeutic opportunity for managing disease progression in patients with DMD.

Authors

Xin Xie, Sophia Y. Tsai, Ming-Jer Tsai