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Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Boston University and Boston Medical Center

Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish β-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with β-globin-expressing cells showing increased maturation. Compared with other cells, β-globin-expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

Induced pluripotent stem cell-based mapping of β-globin expression throughout human erythropoietic development.

Significant delays in the rapid development and distribution of diagnostic testing for SARS-CoV-2 (COVID-19) infection have prevented adequate public health management of the disease, impacting the timely mapping of viral spread and the conservation of personal protective equipment. Furthermore, vulnerable populations, such as those served by the Boston Medical Center (BMC), the largest safety net hospital in New England, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions and substance use disorders, lower health maintenance, unstable housing, and a propensity for rapid community spread, highlighting the urgent need for expedient and reliable in-house testing.

Rapid Implementation of a SARS-CoV-2 Diagnostic Quantitative Real-Time PCR Test with Emergency Use Authorization at a Large Academic Safety Net Hospital.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1;TP63), we track and purify these cells as they first emerge as developmentally immature NKX2-1 lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1/TP63 cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

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.

Taylor Matte

Center for Regenerative Medicine

Boston University and Boston Medical Center

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells