Biology subjects

Desai, T. J.

Publications and source records attributed to Desai, T. J..

5 recordsLinked to original sources

bioRxiv2025

Durable reconstitution of sinonasal epithelium by transplant of CFTR gene corrected airway stem cells

Modulator agents that restore cystic fibrosis transmembrane conductance regulator (CFTR) function have revolutionized outcomes in cystic fibrosis, an incurable multisystem disease. Barriers exist to modulator use, making local CFTR gene and cell therapies attractive, especially in the respiratory tract. We used CRISPR to gene-correct CFTR in upper airway basal stem cells (UABCs) and show durable local engraftment into recipient murine respiratory epithelium. Interestingly, the human cells recapitulate the in vivo organization and differentiation of human sinus epithelium, with little expansion or contraction of the engrafted population over time, while retaining expression of the CFTR transgene. Our results indicate that human airway stem cell transplantation with locoregional restoration of CFTR function is a feasible approach for treating CF and potentially other diseases of the respiratory tract.

bioengineering↗

bioRxiv2022

Alveolar cell fate selection and lifelong maintenance of AT2 cells by FGF signaling

The lungs gas exchange surface comprises thin alveolar type 1 (AT1) cells and cuboidal surfactant-secreting AT2 cells that are corrupted in some of the most common and deadly diseases including adenocarcinoma, emphysema, and SARS/Covid-19. These cells arise from an embryonic progenitor whose development into an AT1 or AT2 cell is thought to be dictated by differential mechanical forces. Here we show the critical determinant is FGF signaling. FGF Receptor 2 (Fgfr2) is expressed in mouse progenitors then restricts to nascent AT2 cells and remains on throughout life. Its ligands are expressed in surrounding mesenchyme and can, in the absence of differential mechanical cues, induce purified, uncommitted E16.5 progenitors to form alveolus-like structures with intermingled AT2 and AT1 cells. FGF signaling directly and cell autonomously specifies AT2 fate; progenitors lacking Fgfr2 in vitro and in vivo exclusively acquire AT1 fate. Fgfr2 loss in AT2 cells perinatally results in reprogramming to AT1 fate, whereas loss or inhibition later in life immediately triggers AT2 apoptosis followed by a compensatory regenerative response. We propose Fgfr2 signaling directly selects AT2 fate during development, induces a cell non-autonomous secondary signal for AT1 fate, and stays on throughout life to continuously maintain healthy AT2 cells. One Sentence SummaryFGF signaling induces and distinguishes the two cell types of the lungs gas exchange surface, and the pathway remains on throughout life to maintain one that can be transformed into lung cancer or targeted in the deadly form of SARS/Covid-19.

developmental biology↗

bioRxiv2021

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

bioengineering↗

bioRxiv2020

Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 "apical-out" organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

cell biology↗

bioRxiv2019

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the {Delta}F508 mutation (found in [~]70% of CF alleles and [~]90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous ({Delta}F508/{Delta}F508) and compound heterozygous ({Delta}F508/Other) CF patients, we achieved 28 {+/-} 5 % and 42 {+/-} 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41{+/-} 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31{+/-} 5 % in UABCs and 51 {+/-} 3 % in HBECs (both from subjects homozygous for {Delta}F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

bioengineering↗