Airway basal cells from human-induced pluripotent stem cells: a new frontier in cystic fibrosis research.

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases, regenerative medicine, and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs, which involves step-by-step differentiation of hiPSCs into definitive endoderm, anterior foregut endoderm, NKX2.1+ lung progenitors, and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs, expressing basal cell markers (NGFR, KRT5, and TP63), could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs, potentially applicable for gene therapy research.

Comparative Study of the Protective and Neurotrophic Effects of Neuronal and Glial Progenitor Cells-Derived Conditioned Media in a Model of Glutamate Toxicity In Vitro.

Cell therapy represents a promising approach to the treatment of neurological diseases, offering potential benefits not only by cell replacement but also through paracrine secretory activities. However, this approach includes a number of limiting factors, primarily related to safety. The use of conditioned stem cell media can serve as an equivalent to cell therapy while avoiding its disadvantages. The present study was a comparative investigation of the antioxidant, neuroprotective and neurotrophic effects of conditioned media obtained from neuronal and glial progenitor cells (NPC-CM and GPC-CM) on the PC12 cell line in vitro. Neuronal and glial progenitor cells were obtained from iPSCs by directed differentiation using small molecules. GPC-CM reduced apoptosis, ROS levels and increased viability, expressions of the antioxidant response genes HMOX1 and NFE2L2 in a model of glutamate-induced oxidative stress. The neurotrophic effect was evidenced by a change in the morphology of pheochromocytoma cells to a neuron-like phenotype. Moreover, neurite outgrowth, expression of GAP43, TUBB3, MAP2, SYN1 genes and increased levels of the corresponding MAP2 and TUBB3 proteins. Treatment with NPC-CM showed moderate antiapoptotic effects and improved cell viability. This study demonstrated the potential application of CM in the field of regenerative medicine.

Comparative study of the efficacy of intra-arterial and intravenous transplantation of human induced pluripotent stem cells-derived neural progenitor cells in experimental stroke.

Background: Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine. Methods: Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed. Results: The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy.

Extracellular vesicles of human glial cells exert neuroprotective effects via brain miRNA modulation in a rat model of traumatic brain injury.

Stem cell-based therapeutic approaches for neurological disorders are widely studied. Paracrine factors secreted by stem cells in vitro and delivered intranasally might allow bypassing the disadvantages associated with a surgical cell delivery procedure with likely immune rejection of a transplant. In this study, we investigated the therapeutic effect of the extracellular vesicles secreted by glial progenitor cells (GPC-EV) derived from human induced pluripotent stem cell in a traumatic brain injury model. Intranasal administration of GPC-EV to Wistar rats for 6 days improved sensorimotor functions assessed over a 14-day observation period. Beside, deep sequencing of microRNA transcriptome of GPC-EV was estimate, and was revealed 203 microRNA species that might be implicated in prevention of various brain pathologies. Modulation of microRNA pools might contribute to the observed decrease in the number of astrocytes that inhibit neurorecovery processes while enhancing neuroplasticity by decreasing phosphorylated Tau forms, preventing inflammation and apoptosis associated with secondary damage to brain tissue. The course of GPC-EV administration was promoted the increasing protein levels of NF-κB in studied areas of the rat brain, indicating NF-κB dependent mechanisms as a plausible route of neuroprotection within the damaged area. This investigation showed that GPC-EV may be representing a therapeutic approach in traumatic brain injury, though its translation into the clinic would require an additional research and development.

Therapeutic Efficiency of Proteins Secreted by Glial Progenitor Cells in a Rat Model of Traumatic Brain Injury.

Traumatic brain injuries account for 30-50% of all physical traumas and are the most common pathological diseases of the brain. Mechanical damage of brain tissue leads to the disruption of the blood-brain barrier and the massive death of neuronal, glial, and endothelial cells. These events trigger a neuroinflammatory response and neurodegenerative processes locally and in distant parts of the brain and promote cognitive impairment. Effective instruments to restore neural tissue in traumatic brain injury are lacking. Glial cells are the main auxiliary cells of the nervous system, supporting homeostasis and ensuring the protection of neurons through contact and paracrine mechanisms. The glial cells' secretome may be considered as a means to support the regeneration of nervous tissue. Consequently, this study focused on the therapeutic efficiency of composite proteins with a molecular weight of 5-100 kDa secreted by glial progenitor cells in a rat model of traumatic brain injury. The characterization of proteins below 100 kDa secreted by glial progenitor cells was evaluated by proteomic analysis. Therapeutic effects were assessed by neurological outcomes, measurement of the damage volume by MRI, and an evaluation of the neurodegenerative, apoptotic, and inflammation markers in different areas of the brain. Intranasal infusions of the composite protein product facilitated the functional recovery of the experimental animals by decreasing the inflammation and apoptotic processes, preventing neurodegenerative processes by reducing the amounts of phosphorylated Tau isoforms Ser396 and Thr205. Consistently, our findings support the further consideration of glial secretomes for clinical use in TBI, notably in such aspects as dose-dependent effects and standardization.

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance.

Airway and lung organoids derived from human-induced pluripotent stem cells (hiPSCs) are current models for personalized drug screening, cell-cell interaction studies, and lung disease research. We analyzed the existing differentiation protocols and identified the optimal conditions for obtaining organoids. In this article, we describe a step-by-step protocol for differentiating hiPSCs into airway and lung organoids. We obtained airway and lung organoids from a healthy donor and from five donors with cystic fibrosis. Analysis of the cellular composition of airway and lung organoids showed that airway organoids contain proximal lung epithelial cells, while lung organoids contain both proximal and distal lung epithelial cells. Forskolin-induced swelling of organoids derived from a healthy donor showed that lung organoids, as well as airway organoids, contain functional epithelial cells and swell after 24 h exposure to forskolin, which makes it a suitable model for analyzing the cystic fibrosis transmembrane conductance regulator (CFTR) channel conductance in vitro. Thus, our results demonstrate the feasibility of generating and characterizing airway and lung organoids from hiPSCs, which can be used for a variety of future applications.

Therapeutic Effects of hiPSC-Derived Glial and Neuronal Progenitor Cells-Conditioned Medium in Experimental Ischemic Stroke in Rats.

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years, and the results are promising. Recent investigations have shown that the administration of the conditioned media obtained after stem cell cultivation can also be effective in the therapy of the central nervous system pathology (hypothesis of their paracrine action). The aim of this study was to evaluate the therapeutic effects of the conditioned medium of hiPSC-derived glial and neuronal progenitor cells in the rat middle cerebral artery occlusion model of the ischemic stroke. Secretory activity of the cultured neuronal and glial progenitor cells was evaluated by proteomic and immunosorbent-based approaches. Therapeutic effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration and the numbers of formed blood vessels in the affected area of the brain. As a result, 31% of the protein species discovered in glial progenitor cells-conditioned medium and 45% in neuronal progenitor cells-conditioned medium were cell type specific. The glial progenitor cell-conditioned media showed a higher content of neurotrophins (BDNF, GDNF, CNTF and NGF). We showed that intra-arterial administration of glial progenitor cells-conditioned medium promoted a faster decrease in neurological deficit compared to the control group, reduced microglia/macrophage infiltration, reduced expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13) and promoted the formation of blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. The results indicate pronounced cytoprotective, anti-inflammatory and angiogenic properties of soluble factors secreted by glial progenitor cells.