Optimizing Nodal, Wnt and BMP signaling pathways for robust and efficient differentiation of human induced pluripotent stem cells to intermediate mesoderm cells.

Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 µM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 µM CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.

Enhanced Immunomodulatory Effects of Thymosin-Alpha-1 in Combination with Polyanionic Carbosilane Dendrimers against HCMV Infection.

Resistance and toxicity associated with current treatments for human cytomegalovirus (HCMV) infection highlight the need for alternatives and immunotherapy has emerged as a promising strategy. This study examined the in vitro immunological effects of co-administration of Thymosin-alpha-1 (Tα1) and polyanionic carbosilane dendrimers (PCDs) on peripheral blood mononuclear cells (PBMCs) during HCMV infection. The biocompatibility of PCDs was assessed via MTT and LDH assays. PBMCs were pre-treated with the co-administered compounds and then exposed to HCMV for 48 h. Morphological alterations in PBMCs were observed using optical microscopy and total dendritic cells (tDCs), myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs), along with CD4+/CD8+ T cells and regulatory T cells (Treg), and were characterized using multiparametric flow cytometry. The findings revealed that Tα1 + PCDs treatments increased DC activation and maturation. Furthermore, increased co-receptor expression, intracellular IFNγ production in T cells and elevated Treg functionality and reduced senescence were evident with Tα1 + G2-S24P treatment. Conversely, reduced co-receptor expression, intracellular cytokine production in T cells, lower functionality and higher senescence in Treg were observed with Tα1 + G2S16 treatment. In summary, Tα1 + PCDs treatments demonstrate synergistic effects during early HCMV infection, suggesting their use as an alternative therapeutic for preventing virus infection.

Persistent Exhausted T-Cell Immunity after Severe COVID-19: 6-Month Evaluation in a Prospective Observational Study.

INTRODUCTION: Severe COVID-19 can result in a significant and irreversible impact on long-term recovery and subsequent immune protection. Understanding the complex immune reactions may be useful for establishing clinically relevant monitoring. METHODS: Hospitalized adults with SARS-CoV-2 between March/October 2020 (n = 64) were selected. Cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained at hospitalization (baseline) and 6 months after recovery. Immunological components' phenotyping and SARS-CoV-2-specific T-cell response were studied in PBMCs by flow cytometry. Up to 25 plasma pro/anti-inflammatory cytokines/chemokines were assessed by LEGENDplex immunoassays. The SARS-CoV-2 group was compared to matched healthy donors. RESULTS: Biochemical altered parameters during infection were normalized at a follow-up time point in the SARS-CoV-2 group. Most of the cytokine/chemokine levels were increased at baseline in the SARS-CoV-2 group. This group showed increased Natural Killer cells (NK) activation and decreased CD16high NK subset, which normalized six months later. They also presented a higher intermediate and patrolling monocyte proportion at baseline. T cells showed an increased terminally differentiated (TemRA) and effector memory (EM) subsets distribution in the SARS-CoV-2 group at baseline and continued to increase six months later. Interestingly, T-cell activation (CD38) in this group decreased at the follow-up time point, contrary to exhaustion markers (TIM3/PD1). In addition, we observed the highest SARS-CoV-2-specific T-cell magnitude response in TemRA CD4 T-cell and EM CD8 T-cell subsets at the six-months time point. CONCLUSIONS: The immunological activation in the SARS-CoV-2 group during hospitalization is reversed at the follow-up time point. However, the marked exhaustion pattern remains over time. This dysregulation could constitute a risk factor for reinfection and the development of other pathologies. Additionally, high SARS-CoV-2-specific T-cells response levels appear to be associated with infection severity.

Effects of Hypocalcemic Vitamin D Analogs in the Expression of DNA Damage Induced in Minilungs from hESCs: Implications for Lung Fibrosis.

In our previous work, we evaluated the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of bleomycin-induced lung fibrosis. Contrary to the expected, vitamin D supplementation increased the DNA damage expression and cellular senescence in alveolar epithelial type II cells and aggravated the overall lung pathology induced in mice by bleomycin. These effects were probably due to an alteration in the cellular DNA double-strand breaks' repair capability. In the present work, we have evaluated the effects of two hypocalcemic vitamin D analogs (calcipotriol and paricalcitol) in the expression of DNA damage in the context of minilungs derived from human embryonic stem cells and in the cell line A549.

The Role of BMP Signaling in Female Reproductive System Development and Function.

Bone morphogenetic proteins (BMPs) are a group of multifunctional growth factors that belong to the transforming growth factor-ß (TGF-ß) superfamily of proteins. Originally identified by their ability to induce bone formation, they are now known as essential signaling molecules that regulate the development and function of the female reproductive system (FRS). Several BMPs play key roles in aspects of reproductive system development. BMPs have also been described to be involved in the differentiation of human pluripotent stem cells (hPSCs) into reproductive system tissues or organoids. The role of BMPs in the reproductive system is still poorly understood and the use of FRS tissue or organoids generated from hPSCs would provide a powerful tool for the study of FRS development and the generation of new therapeutic perspectives for the treatment of FRS diseases. Therefore, the aim of this review is to summarize the current knowledge about BMP signaling in FRS development and function.

Detrimental pro-senescence effects of vitamin D on lung fibrosis.

BACKGROUND: The multiple biological effects of vitamin D and its novel activities on inflammation and redox homeostasis have raised high expectations on its use as a therapeutic agent for multiple fibrogenic conditions. We have assessed the therapeutic effects of 1α,25-Dihydroxyvitamin D3, the biologically active form of vitamin D, in the context of lung fibrosis. METHODS: We have used representative cellular models for alveolar type II cells and human myofibroblasts. The extension of DNA damage and cellular senescence have been assessed by immunofluorescence, western-blot and senescence-associated ß-galactosidase activity. We have also set up a murine model for lung fibrosis by intraperitoneal injections of bleomycin. RESULTS: Vitamin D induces cellular senescence in bleomycin-treated alveolar epithelial type II cells and aggravates the lung pathology induced by bleomycin. These effects are probably due to an alteration of the cellular DNA double-strand breaks repair in bleomycin-treated cells. CONCLUSIONS: The detrimental effects of vitamin D in the presence of a DNA damaging agent might preclude its use as an antifibrogenic agent for pulmonary fibrosis characterized by DNA damage occurrence and cellular senescence.

Effects of lung and airway epithelial maturation cocktail on the structure of lung bud organoids.

Organoids from human pluripotent stem cells are becoming suitable models for studies of organ development, drug screening, regenerative medicine, and disease modeling. Three-dimensional minilungs in Matrigel culture have recently been generated from human embryonic stem cells. These particular organoids, named lung bud organoids, showed branching airway and early alveolar structures resembling those present in lungs from the second trimester of human gestation. We show here that the treatment of such organoids with a lung and airway epithelial maturation cocktail containing dexamethasone drives lung bud organoids to the formation of paddle-racquet like structures. This strategy may help to increase the versatility of lung organoids and to generate structures more advanced than the original branching texture.

A Two-Dimensional Human Minilung System (Model) for Respiratory Syncytial Virus Infections.

Human respiratory syncytial virus (HRSV) is a major cause of serious pediatric respiratory diseases that lacks effective vaccine or specific therapeutics. Although our understanding about HRSV biology has dramatically increased during the last decades, the need for adequate models of HRSV infection is compelling. We have generated a two-dimensional minilung from human embryonic stem cells (hESCs). The differentiation protocol yielded at least six types of lung and airway cells, although it is biased toward the generation of distal cells. We show evidence of HRSV replication in lung cells, and the induction of innate and proinflammatory responses, thus supporting its use as a model for the study of HRSV-host interactions.