Center for
regenerative medicine

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…

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Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and can give rise to all the mature blood cell types in our body, while at the same time maintaining a pool of HSCs through self-renewing divisions. This potential is reflected in their functional definition as cells that are capable of long-term multi-lineage engraftment upon transplantation. While all HSCs meet these criteria, subtle differences exist between developmentally different populations of these cells. Here we present a comprehensive overview of traditional and more recently described markers for phenotyping HSCs and their downstream progeny. To address the need to assess the growing number of surface molecules expressed in various HSC-enriched fractions at different developmental stages, we have developed an extensive multi-parameter spectral flow cytometry panel to phenotype hematopoietic stem and multipotent progenitor cells (HSC/MPPs) throughout development. In this study we then employ this panel to comprehensively profile the HSC compartment in the human fetal liver (FL), which is endowed with superior engraftment potential compared to postnatal sources. Spectral cytometry lends an improved resolution of marker expression to our comprehensive approach, allowing to extract combinatorial expression signatures of several relevant HSC/MPP markers to precisely characterize the HSC/MPP fraction in a variety of tissues.

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Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor–derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide “proof of principle” that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.

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There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

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The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

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Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial to hematopoietic transition (EHT) to give rise to hematopoietic progenitors. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs), but lung HECs have never been described. Using explant cultures of murine and human fetal lungs, we demonstrate that the fetal lung is a source of HECs that have the functional capacity to undergo EHT to produce de-novo HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis.

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Individuals homozygous for the pathogenic “Z” mutation in alpha-1 antitrypsin deficiency (AATD) are known to be at increased risk for chronic liver disease. That some degree of risk is similarly conferred by the heterozygous state, estimated to affect 2% of the US population, has also become clear. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of expressing a single ZAAT allele on hepatocyte biology. Here, through the application of CRISPR-Cas9 editing, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-derived hepatocytes (iHeps) relative to homozygous mutant (ZZ) or corrected (MM) cells. We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations in hepatic metabolic function including ER and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in subpopulations of cells. Our cellular model of MZ heterozygosity thus provides evidence that expression of a single Z allele is sufficient to disrupt hepatocyte homeostatic function and suggest a mechanism underlying the increased risk of liver disease observed among MZ individuals.

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