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
Lung alveolar type 2 (AT2) cells are progenitors for alveolar type 1 (AT1) cells. Although many factors regulate AT2 cell plasticity, the role of mitochondrial calcium (mCa2+) uptake in controlling AT2 cells remains unclear. We previously identified that the microRNA family, miR-302, supports lung epithelial progenitor cell proliferation and less differentiated phenotypes during development. Here we report that a sustained elevation of miR-302 in adult AT2 cells decreases AT2-to-AT1 cell differentiation during the Streptococcus pneumoniae induced lung injury repair. We identified that miR-302 targets and represses the expression of mitochondrial Ca2+ uptake 1 (MICU1), which regulates mCa2+ uptake through the mCa2+ uniporter channel by acting as a gatekeeper at low cytosolic Ca2+ levels. Our results reveal a marked increase in MICU1 protein expression and decreased mCa2+ uptake during AT2-to-AT1 cell differentiation in the adult lung. Deletion of Micu1 in AT2 cells reduces AT2-to-AT1 cell differentiation during steady-state tissue maintenance and alveolar epithelial regeneration following bacterial pneumonia. These studies indicate that mCa2+ uptake is extensively modulated during AT2-to-AT1 cell differentiation and that MICU1-dependent mCa2+ uniporter channel gating is a prominent mechanism modulating AT2-to-AT1 cell differentiation.
Mir Ali, Xiaoying Zhang, Ryan LaCanna, Dhanendra Tomar, John W. Elrod, Ying Tian
Spiral ganglion neurons (SGNs) are primary auditory neurons in the spiral ganglion that transmit sound information from the inner ear to the brain and play an important role in hearing. Impairment of SGNs causes sensorineural hearing loss (SNHL), and it has been thought until now that SGNs cannot be regenerated once lost. Furthermore, no fundamental therapeutic strategy for SNHL has been established other than inserting devices such as hearing aids and cochlear implants. Here we show that the mouse spiral ganglion contains cells that are able to proliferate and indeed differentiate into neurons in response to injury. We suggest that SRY-box transcription factor 2/SRY-box transcription factor 10–double-positive (Sox2/Sox10–double-positive) Schwann cells sequentially started to proliferate, lost Sox10 expression, and became neurons, although the number of new neurons generated spontaneously was very small. To increase the abundance of new neurons, we treated mice with 2 growth factors in combination with valproic acid, which is known to promote neuronal differentiation and survival. This treatment resulted in a dramatic increase in the number of SGNs, accompanied by a partial recovery of the hearing loss induced by injury. Taken together, our findings offer a step toward developing strategies for treatment of SNHL.
Takahiro Wakizono, Hideyuki Nakashima, Tetsuro Yasui, Teppei Noda, Kei Aoyagi, Kanako Okada, Yasuhiro Yamada, Takashi Nakagawa, Kinichi Nakashima
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