The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR–dependent manner.
Isaac M. Oderberg, Wolfram Goessling
Keratin expression dynamically changes in airway basal cells (BCs) following acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In Bronchiolitis Obliterans (BO) following lung transplantation, BC clonogenicity declines which is associated with a switch from Keratin15 (Krt15) to Keratin14 (Krt14). We investigated the roles of these keratins using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs failed to differentiate into club and ciliated cells, but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation, but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo following injury. Krt14, but not Krt15, bound the tumor suppressor Stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, while dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts that of Krt14-KO and resembles the phenotype in BO with decreased clonogenicity, increased Krt14 and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior which is relevant in chronic disease states like BO.
Vitaly Ievlev, Thomas J. Lynch, Kyle W. Freischlag, Caitlyn B. Gries, Anit Shah, Albert C. Pai, Bethany A. Ahlers, Soo Yeun Park, John F. Engelhardt, Kalpaj R. Parekh
Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we perform head-to-head comparisons between the transcriptomes of fresh primary (1o) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1o and iAEC2s) exhibiting similarities to and differences from freshly purified 1o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires a “transitional cell state” described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1o and engineered AEC2s, two in vitro models that can be harnessed to study human lung health and disease.
Konstantinos-Dionysios Alysandratos, Carolina Garcia-de-Alba, Changfu Yao, Patrizia Pessina, Jessie Huang, Carlos Villacorta-Martin, Olivia T. Hix, Kasey Minakin, Claire L. Burgess, Pushpinder Bawa, Aditi Murthy, Bindu Konda, Michael F. Beers, Barry R. Stripp, Carla F. Kim, Darrell N. Kotton
Diamond–Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.
Senthil Velan Bhoopalan, Jonathan S. Yen, Thiyagaraj Mayuranathan, Kalin D. Mayberry, Yu Yao, Maria Angeles Lillo Osuna, Yoonjeong Jang, Janaka S.S. Liyange, Lionel Blanc, Steven R. Ellis, Marcin W. Wlodarski, Mitchell J. Weiss
Individuals with beta-thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the gamma-globin promoters in “healthy” CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in >60% -113A>G conversion with 30% γ-globin of human beta globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from beta-thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.
Chang Li, Aphrodite Georgakopoulou, Gregory A. Newby, Kelcee A. Everette, Evangelos Nizamis, Kiriaki Paschoudi, Efthymia Vlachaki, Sucheol Gil, Anna K. Anderson, Theodore Koob, Lishan Huang, Hongjie Wang, Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, André Lieber
Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex inhibitors (GSC), neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent post-translational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. The GSC and glycogen synthase kinase 3 were implicated in these post-translational events but WNT agonist/antagonist studies and RNA sequencing indicated a WNT-independent mechanism. Collectively, these data suggest that post-translational modifications create distinct assemblies of JAG1 and JAG2 which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.
Susan D. Reynolds, Cynthia L. Hill, Alfahdah Alsudayri, Scott W. Lallier, Saranga Wijeratne, Zheng Hong Tan, Tendy Chiang, Estelle Cormet-Boyaka
Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.
Sydney Felker, Archana Shrestha, Jeff Bailey, Devin M Pillis, Dylan Siniard, Punam Malik
Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell–derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.
Kristine M. Abo, Julio Sainz de Aja, Jonathan Lindstrom-Vautrin, Konstantinos-Dionysios Alysandratos, Alexsia Richards, Carolina Garcia-de-Alba, Jessie Huang, Olivia T. Hix, Rhiannon B. Werder, Esther Bullitt, Anne Hinds, Isaac Falconer, Carlos Villacorta-Martin, Rudolf Jaenisch, Carla F. Kim, Darrell N. Kotton, Andrew A. Wilson
Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.
Stefan Klingler, Kuo-Shun Hsu, Guoqiang Hua, Maria Laura Martin, Mohammad Adileh, Timour Baslan, Zhigang Zhang, Philip B. Paty, Zvi Fuks, Anthony M.C. Brown, Richard Kolesnick
Chronic myeloproliferative neoplasms (MPN) frequently evolve to a blast phase (BP) that is almost uniformly resistant to induction chemotherapy or hypomethylating agents. We explored the functional properties, genomic architecture, and cell of origin of MPN-BP initiating cells (IC) using a serial NSG mouse xenograft transplantation model. Transplantation of peripheral blood mononuclear cells (MNC) from 7 of 18 patients resulted in a high degree of leukemic cell chimerism and re-created clinical characteristics of human MPN-BP. The function of MPN-BP ICs was not dependent on the presence of JAK2V617F, a driver mutation associated with the initial underlying MPN. By contrast, multiple MPN-BP IC subclones co-existed within MPN-BP MNCs characterized by different myeloid malignancy gene mutations and cytogenetic abnormalities. MPN-BP ICs in 4 patients exhibited extensive proliferative and self-renewal capacity as demonstrated by their ability to recapitulate human MPN-BP in serial recipients. These MPN-BP IC subclones underwent extensive continuous clonal competition within individual xenografts and across multiple generations and their subclonal dynamics were consistent with functional evolution of MPN-BP IC. Finally, we show that MPN-BP ICs originate from not only phenotypically identified hematopoietic stem cells but also lymphoid-myeloid progenitor cells which were each characterized by differences in MPN-BP initiating activity and self-renewal capacity.
Xiaoli Wang, Raajit K. Rampal, Cing Siang Hu, Joseph Tripodi, Noushin Farnoud, Bruce Petersen, Michael R. Rossi, Minal Patel, Erin McGovern, Vesna Najfeld, Camelia Iancu-Rubin, Min Lu, Andrew Davis, Marina Kremyanskaya, Rona Singer Weinberg, John Mascarenhas, Ronald Hoffman
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