Finn Hawkins

Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells

Two populations of Nkx2-1(+) progenitors in the developing foregut endoderm give rise to the entire postnatal lung and thyroid epithelium, but little is known about these cells because they are difficult to isolate in a pure form. We demonstrate here the purification and directed differentiation of primordial lung and thyroid progenitors derived from mouse embryonic stem cells (ESCs). Inhibition of TGFβ and BMP signaling, followed by combinatorial stimulation of BMP and FGF signaling, can specify these cells efficiently from definitive endodermal precursors. When derived using Nkx2-1(GFP) knockin reporter ESCs, these progenitors can be purified for expansion in culture and have a transcriptome that overlaps with developing lung epithelium. Upon induction, they can express a broad repertoire of markers indicative of lung and thyroid lineages and can recellularize a 3D lung tissue scaffold. Thus, we have derived a pure population of progenitors able to recapitulate the developmental milestones of lung/thyroid development.

Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

Summary Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Regeneration of Thyroid Function by Transplantation of Differentiated Pluripotent Stem Cells

Differentiation of functional thyroid epithelia from pluripotent stem cells (PSCs) holds the potential for application in regenerative medicine. However, progress toward this goal is hampered by incomplete understanding of the signaling pathways needed for directed differentiation without forced overexpression of exogenous transgenes. Here we use mouse PSCs to identify key conserved roles for BMP and FGF signaling in regulating thyroid lineage specification from foregut endoderm in mouse and Xenopus. Thyroid progenitors derived from mouse PSCs can be matured into thyroid follicular organoids that provide functional secretion of thyroid hormones in vivo and rescue hypothyroid mice after transplantation. Moreover, by stimulating the same pathways, we were also able to derive human thyroid progenitors from normal and disease-specific iPSCs generated from patients with hypothyroidism resulting from NKX2-1 haploinsufficiency. Our studies have therefore uncovered the regulatory mechanisms that underlie early thyroid organogenesis and provide a significant step toward cell-based regenerative therapy for hypothyroidism.

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

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 NKX2-1GFP 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 CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

Pluripotent stem cell differentiation reveals distinct developmental pathways regulating lung versus thyroid lineage specification

The in vitro-directed differentiation of pluripotent stem cells (PSCs) through stimulation of developmental signaling pathways can generate mature somatic cell types for basic laboratory studies or regenerative therapies. However, there has been significant uncertainty regarding a method to separately derive lung versus thyroid epithelial lineages, as these two cell types each originate from Nkx2-1+ foregut progenitors and the minimal pathways claimed to regulate their distinct lineage specification in vivo or in vitro have varied in previous reports. Here, we employ PSCs to identify the key minimal signaling pathways (Wnt+BMP versus BMP+FGF) that regulate distinct lung- versus thyroid-lineage specification, respectively, from foregut endoderm. In contrast to most previous reports, these minimal pathways appear to be evolutionarily conserved between mice and humans, and FGF signaling, although required for thyroid specification, unexpectedly appears to be dispensable for lung specification. Once specified, distinct Nkx2-1+ lung or thyroid progenitor pools can now be independently derived for functional 3D culture maturation, basic developmental studies or future regenerative therapies. Summary: Delineation of the minimal evolutionarily conserved signaling pathways required for generating lung or thyroid progenitors from pluripotent stem cells aids the provision of an inexhaustible source of progenitors for further investigations.

Keeping it in the family: three relatives with HbSC disease and simultaneous acute pulmonary emboli.

A 30-year-old African American female (Patient #1) with Hemoglobin SC (HbSC) disease presented with a 1 week history of progressive left flank and lower extremity pain with diffuse myalgias. She had a history of multiple prior vasoocclusive episodes requiring simple and exchange transfusions. At the time of admission on physical examination, she was afebrile and hemodynamically stable with an oxygen saturation of 100% in ambient air. The only pertinent finding was mild diffuse abdominal tenderness. Laboratory studies are summarized in Table 1 and were significant for leukocytosis with a left shift, anemia worse than baseline, a depressed reticulocyte count, a normal bilirubin but a mild elevation in transaminases. Chest radiography was normal. HbSC disease is a compound heterozygous condition characterized by inheritance of one gene for ‘‘S’’ hemoglobin (Glu-Val substitution in the 6th position of the b-globin chain) and one gene for ‘‘C’’ hemoglobin (Glu-Lys substitution in the 6th position of the b-globin chain), with subsequent formation of similar levels of HbS and HbC within the erythrocyte. HbCC disease is a non-sickling disorder which results in a mild hemolytic anemia. The combination of HbS and HbC is much more clinically significant suggesting that HbS has major disease modifying effect. Population studies suggest that while patients with HbSC have less frequent complications occurring later in life than those with HbSS disease, when complications occur they can be equally severe. In contrast, there is generally milder anemia and hemolytic complications, including aplastic episodes and cholelithiasis are less severe compared to HbSS disease [1]. In our first patient, the presence of diffuse pain was suggestive of a vasoocclusive episode (VOE). There were no abnormalities on history, physical exam, or chest radiography suggestive of acute chest syndrome (ACS). In terms of anemia, this patient was noted to have approximately a 3.5 g decrease in Hb concentration with an inappropriately low reticulocyte response. This suggested a VOE complicated by an aplastic crisis. Patient #1 was admitted to the medical service and commenced on treatment for a vasoocclusive epsiode with intravenous fluids and analgesics. She was commenced on low molecular weight heparin (LMWH) for deep venous thrombosis (DVT) prophylaxis. A red blood cell transfusion was administered for treatment of symptomatic anemia. The next day, the 24-year-old brother of Patient #1 (Patient #2), was admitted to the medical service. He also had HbSC disease and lived in same household as Patient #1. He had a history of cough productive of green sputum, fever, chills, myalgia and lower back pain for one week prior to admission. On physical examination, he was afebrile and had an oxygen saturation of 100% in ambient air. The rest of his exam was unremarkable other than pain with limb and spine motion. Laboratory studies are summarized in Table 1 and were significant for a leukocytosis with a left shift, worse anemia from baseline, an abnormally reduced reticulocyte response and mild hyperbilirubinemia. Chest radiography was normal. He was treated with intravenous fluids, analgesics and was administered LMWH for DVT prophylaxis. One of the recognized complications of sickle cell disease (SCD) is a reticulocytopenic anemia, referred to as an aplastic crisis or episode. This condition can be associated with a decrease in any peripheral blood cell lines, but most commonly, there are decreased mature erythrocytes and reticulocytes, and this is referred to as transient red cell aplasia (TRCA) [2]. A number of infectious agents have been implicated as either being causative or temporally related to such an episode. The best detailed of these is Parvovirus B19 (HPV B19), the causative agent of fifth disease. This virus is the only member of the Parvoviridae family known to be pathogenic in humans, occurring worldwide endemically [3]. Most infections occur in children and young adults and are highly contagious. The association between parvovirus infection and hypoplastic episodes in sickle cell anemia was first identified in 1981, when sera from 600 children in a London hospital were analyzed for HPV B19. Six children were identified with either the presence of the B19 antigen or its acute phase antibody [4]. All six were originally from Jamaica, had SCD with worsening anemia and evidence of bone marrow erythroid hypoplasia [4]. Other viruses recognized to be associated with red cell aplasia, although not specifically in SCD, include Epstein Barr, mumps, hepatitis, and Human Herpes virus 6 [5,6].

Single-Cell Transcriptomic Profiling of Pluripotent Stem Cell-Derived SCGB3A2+ Airway Epithelium

Summary Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.

Pulmonary Ionocytes Challenge the Paradigm in Cystic Fibrosis

Two recent studies have identified novel airway cells termed pulmonary ionocytes that express higher levels of CFTR than other airway cells express. These findings raise new questions in the evolving debate about the physiological role of CFTR in lung epithelia and its importance in the pathogenesis of cystic fibrosis (CF).

Derivation of Epithelial‐Only Airway Organoids from Human Pluripotent Stem Cells

New protocols to efficiently generate functional airway epithelial organoids from human pluripotent stem cells (PSCs) would represent a major advance towards effective disease modeling, drug screening and cell based therapies for lung disorders. This unit describes an approach using stage-specific signaling pathway manipulation to differentiate cells to proximal airway epithelium via key developmental intermediates. Cells are directed via definitive endoderm (DE) to anterior foregut, and then specified to NKX2-1+ lung epithelial progenitors. These lung progenitors are purified using cell surface marker sorting and replated in defined culture conditions to form three-dimensional, epithelial-only airway organoids. This directed differentiation approach using serum-free, defined media also includes protocols for evaluation of DE induction, intracellular FACS analysis of NKX2-1 specification efficiency and enrichment, and approaches for characterization and expansion of airway organoids. Taken together, this represents an efficient and reproducible approach to generate expandable airway organoids from human PSCs for use in numerous downstream applications.

The Genetic Programs Regulating Embryonic Lung Development and Induced Pluripotent Stem Cell Differentiation

This chapter reviews the current knowledge of the molecular mechanisms controlling embryonic lung development in animal models from the initial specification of a small number of respiratory progenitor cells in the ventral foregut endoderm through the formation of the mature adult lung with regionally specialized epithelial, interstitial, and vascular compartments. In the second half of this chapter we introduce induced pluripotent stem cells (iPSCs) as a compelling new platform to study human lung biology at developmental time-points previously inaccessible to researchers. iPSCs offer the potential to generate functional lung tissue in vitro by translating the knowledge gained from studying respiratory system development in different animal models where many of the signaling pathways or airway branching mechanisms are evolutionarily conserved. There are many exciting possible applications of iPSC-derived lung tissue, including the ability to model human lung disease, screen novel drug therapies, and ultimately generate functional, transplantable lung cells or 3-D tissues for those suffering from one of the many forms of end-stage lung disease.