chemogenetic-regulation-reprogrammed-stem-cell-derived-precursor

Chemogenetic Regulation in Reprogrammed Stem Cell-derived Precursor Cells in Treating Neurodegenerative Diseases

The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for ...

Xuanwu Hospital Capital Medical University

Ministry of Education

Aggregation of α-Synuclein, possibly caused by disturbance of proteostasis, has been identified as a common pathological feature of Parkinson's disease (PD). However, the initiating events of aggregation have not been fully illustrated, and this knowledge may be critical to understanding the disease mechanisms of PD. Proteostasis is essential in maintaining normal cellular metabolic functions, which regulate the synthesis, folding, trafficking, and degradation of proteins. The toxicity of the aggregating proteins is dramatically influenced by its physical and physiological status. Genetic mutations may also affect the metastable phase transition of proteins. In addition, neuroinflammation, as well as lipid metabolism and its interaction with α-Synuclein, are likely to contribute to the pathogenesis of PD. In this review article, we will highlight recent progress regarding α-Synuclein proteostasis in the context of PD. We will also discuss how the phase transition status of α-Synuclein could correlate with different functional consequences in PD.

Proteostasis of α-Synuclein and Its Role in the Pathogenesis of Parkinson's Disease.

Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells that can be derived from various tissues. Due to their regenerative and immunomodulatory properties, MSCs have been extensively researched and tested for treatment of different diseases/indications. One mechanism that MSCs exert functions is through the transfer of mitochondria, a key player involved in many biological processes in health and disease. Mitochondria transfer is bidirectional and has an impact on both donor and recipient cells. In this review, we discussed how MSC-mediated mitochondrial transfer may affect cellular metabolism, survival, proliferation, and differentiation; how this process influences inflammatory processes; and what is the molecular machinery that mediates mitochondrial transfer. In the end, we summarized recent advances in preclinical research and clinical trials for the treatment of stroke and spinal cord injury, through application of MSCs and/or MSC-derived mitochondria.

Mesenchymal Stem/Stromal Cell-Mediated Mitochondrial Transfer and the Therapeutic Potential in Treatment of Neurological Diseases.

Barrett's esophagus is a major complication of gastro-esophageal reflux disease and an important precursor lesion for the development of Barrett's metaplasia and esophageal adenocarcinoma. However, the cellular and molecular mechanisms of Barrett's metaplasia remain unclear. Inflammation-associated oxidative DNA damage could contribute to Barrett's esophagus. It has been demonstrated that poly(ADP-ribose) polymerases (PARPs)-associated with ADP-ribosylation plays an important role in DNA damage and inflammatory response. A previous study indicated that there is inflammatory infiltration and oxidative DNA damage in the lower esophagus due to acid/bile reflux, and gastric acid could induce DNA damage in culture esophageal cells. This review will discuss the mechanisms of Barrett's metaplasia and adenocarcinoma underlying oxidative DNA damage in gastro-esophageal reflux disease patients based on recent clinical and basic findings.

The Oxidative Damage and Inflammation Mechanisms in GERD-Induced Barrett's Esophagus.

Induced neural stem cells (iNSCs) can be reprogrammed from somatic cells and have shown potentials in treatment of various neurological diseases/disorders. Obtaining iNSCs of nonhuman primates serves as an important bridge for clinical translation using iNSCs. In the current study, cynomolgus (Macaca fascicularis) bone marrow mesenchymal stromal cells (MSCs) were reprogrammed into iNSCs by transduction of non-integrative Sendai virus encoding transgenes OCT4, SOX2, KLF4 and C-MYC. The obtained iNSCs showed characteristics of normal neural stem cells (NSCs) and could differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, iNSCs could give rise to dopaminergic neural cells in vitro, which showed safety and efficacy after transplantation into the striatum of an immunodeficient mouse Parkinson's disease (PD) model.

Induced neural stem cells from Macaca fascicularis show potential of dopaminergic neuron specification and efficacy in a mouse Parkinson's disease model.

Mesenchymal stromal cells (MSCs) exhibit beneficial anti-inflammatory effects against Parkinson's disease (PD) via immunomodulatory actions. However, the underlying molecular mechanism remains unclear. This study aimed to investigate the potential neuroprotective effects of MSCs and the possible mechanisms involved by infusing human umbilical cord MSCs (hMSCs) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mature male C57BL/6 mice. Subsequently, the striatal content of dopamine (DA) and its metabolites, tyrosine hydroxylase (TH)-positive neurons and activated microglia, circulating inflammatory cytokines, gene expression of cytokines, and NOD-like receptor protein 3 (NLRP3) inflammasome molecules were measured using high-performance liquid chromatography, flow cytometry, immunohistochemistry, immunofluorescent staining, and real-time polymerase chain reaction assays, respectively. Infused hMSCs markedly ameliorated the reduction in striatal DA and loss of TH-positive neurons in the substantia nigra of PD mice. The MPTP-induced activation of microglia and increase in tumor necrosis factor-α and interleukin-1β mRNA expression in the striatum were also attenuated by hMSCs. Furthermore, hMSCs completely reversed the elevated pro-inflammatory cytokine levels in the serum and mRNA expression of cytokines in the peripheral organs of PD mice. Moreover, hMSCs significantly inhibited the expression of caspase-1 and NLRP3 of the NLRP3 inflammasome in both the central and peripheral organs at mRNA level. These data suggest that hMSCs protect dopaminergic neurons in PD mice by suppressing both the central and peripheral inflammatory responses, probably by inhibiting the NLRP3 inflammasome. However, the animals in our study only received several MSC infusions, and the effects of infused MSCs over extended periods on the NLRP3 inflammasome need to be investigated in future studies.

Neuroprotective effects of human umbilical cord mesenchymal stromal cells in PD mice via centrally and peripherally suppressing NLRP3 inflammasome-mediated inflammatory responses.

Neural stem cells (NSCs) and derivatives are potential cellular sources to treat neurological diseases. In the current study, we reprogrammed human peripheral blood mononuclear cells into induced NSCs (iNSCs) and inserted GFP gene into the AAVS1 site for graft tracing. Targeted integration of GFP does not affect the proliferation and differentiation capacity of iNSCs. iNSC-GFP can be further differentiated into dopaminergic precursors (DAPs) and motor neuron precursors (MNPs), respectively. iNSCs were engrafted into the motor cortex and iNSC-DAPs into the striatum and substantia nigra (SN) of a nonhuman primate, respectively. The surviving iNSCs could respond to the microenvironment of the cortex and spontaneously differentiate into mature neurons that extended neurites. iNSC-DAPs survived well and matured into DA neurons following transplantation into the striatum and SN. iNSC-MNPs could also survive and turn into motor neurons after being engrafted into the spinal cord of rats. The results suggest that iNSCs and derivatives have a potential to be used for the treatment of neurological diseases.

Characterization of Human-Induced Neural Stem Cells and Derivatives following Transplantation into the Central Nervous System of a Nonhuman Primate and Rats.

Systemic immunomodulation is increasingly recognized among the beneficial effects of mesenchymal stromal cells (MSCs) in treatment of Parkinson's disease (PD), while the underlying mechanism is not fully understood. With the growing popularity of using probiotics as an adjuvant approach in PD treatment, concerns about the added effects of probiotics have been raised. In addition to the molecular mechanism mediating the neuroprotective effects of MSCs, the combined effects of a probiotic formulation, VSL#3, and MSC infusion were also evaluated in PD mice. The animals were weekly treated with human MSCs (hMSCs) via the tail vein, VSL#3 via the gastrointestinal tract, or their combination six times. hMSCs, VSL#3 alone, and their combination markedly ameliorated the decreased striatal dopamine content, loss of dopaminergic neurons in the substantia nigra, increased levels of proinflammatory cytokines in serum, as well as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) mRNAs in striatum and peripheral tissues induced by MPTP. Furthermore, hMSCs, VSL#3, and their combination notably downregulated mRNA expression of NOD-like receptor protein 3 (NLRP3) and caspase-1 in brain and peripheral tissues of PD mice. These results suggest that hMSCs, VSL#3, and their combination prevent neurodegenerative changes in PD mice via anti-inflammatory activities in both the central and peripheral systems, possibly through suppressing the NLRP3 inflammasome. Moreover, two-way analysis of variance (ANOVA) indicated that VSL#3 interacts with hMSCs to attenuate neurodegeneration and inhibit NLRP3 inflammasome-mediated inflammation without altering the effects of hMSCs. Major findings of our study support the usage of probiotic formulation VSL#3 as an adjuvant therapy to hMSC infusion in PD treatment.  This study provides evidence for the neuroprotective activities of human umbilical cord MSCs from the aspect of anti-inflammation actions. hMSCs inhibit the NLRP3 inflammasome and MPTP-induced inflammation in both brain and periphery to relieve the degenerative changes in dopaminergic neurons in PD mice. Furthermore, as an additional therapeutic agent, probiotic formulation VSL#3 interacts with hMSCs in suppressing the NLRP3 inflammasome as well as the central and peripheral anti-inflammatory effects to exert neuroprotective actions in PD mice without altering the actions of hMSCs, suggesting the potential of VSL#3 as an adjuvant therapy in PD treatment. The findings of the present study give a further understanding of the anti-inflammatory activity and the molecular mechanism for the beneficial effects of MSCs as well as the potential application of probiotic formulation as an adjuvant approach to MSC therapy in PD treatment.

Probiotic Formulation VSL#3 Interacts with Mesenchymal Stromal Cells To Protect Dopaminergic Neurons via Centrally and Peripherally Suppressing NOD-Like Receptor Protein 3 Inflammasome-Mediated Inflammation in Parkinson's Disease Mice.

To investigate the efficacy and mechanisms of human umbilical cord-derived MSC-derived extracellular vesicles (hucMSC-EVs) in a mouse model of desiccation-induced dry eye disease (DED).

MicroRNAs of extracellular vesicles derived from mesenchymal stromal cells alleviate inflammation in dry eye disease by targeting the IRAK1/TAB2/NF-κB pathway.

 Parkinson's disease (PD) is a prevalent neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons (DA) at the substantia nigra pas compacta (SNpc). Cell therapy has been proposed as a potential treatment option for PD, with the aim of replenishing the lost DA neurons and restoring motor function. Fetal ventral mesencephalon tissues (fVM) and stem cell-derived DA precursors cultured in 2-dimentional (2-D) culture conditions have shown promising therapeutic outcomes in animal models and clinical trials. Recently, human induced pluripotent stem cells (hiPSC)-derived human midbrain organoids (hMOs) cultured in 3-dimentional (3-D) culture conditions have emerged as a novel source of graft that combines the strengths of fVM tissues and 2-D DA cells.  3-D hMOs were induced from three distinct hiPSC lines. hMOs at various stages of differentiation were transplanted as tissue pieces into the striatum of naïve immunodeficient mouse brains, with the aim of identifying the most suitable stage of hMOs for cellular therapy. The hMOs at Day 15 were determined to be the most appropriate stage and were transplanted into a PD mouse model to assess cell survival, differentiation, and axonal innervation . Behavioral tests were conducted to evaluate functional restoration following hMO treatment and to compare the therapeutic effects between 2-D and 3-D cultures. Rabies virus were introduced to identify the host presynaptic input onto the transplanted cells.  hMOs showed a relatively homogeneous cell composition, mostly consisting of dopaminergic cells of midbrain lineage. Analysis conducted 12 weeks post-transplantation of day 15 hMOs revealed that 14.11% of the engrafted cells expressed TH+ and over 90% of these cells were co-labeled with GIRK2+, indicating the survival and maturation of A9 mDA neurons in the striatum of PD mice. Transplantation of hMOs led to a reversal of motor function and establishment of bidirectional connections with natural brain target regions, without any incidence of tumor formation or graft overgrowth.  The findings of this study highlight the potential of hMOs as safe and efficacious donor graft sources for cell therapy to treat PD.

Human iPSC-derived midbrain organoids functionally integrate into striatum circuits and restore motor function in a mouse model of Parkinson's disease.

Stem cell-based therapy is a promising strategy for treating Parkinson's disease (PD) characterized by the loss of dopaminergic neurons. Recently, induced neural stem cell-derived dopaminergic precursor cells (iNSC-DAPs) have been emerged as a promising candidate for PD cell therapy because of a lower tumor-formation ability. Designer receptors exclusively activated by designer drugs (DREADDs) are useful tools for examining functional synaptic connections with host neurons.

Modulation of human induced neural stem cell-derived dopaminergic neurons by DREADD reveals therapeutic effects on a mouse model of Parkinson's disease.

Cell transplantation is a promising approach for addressing neurodegenerative conditions. In this study, we developed a robot-assisted stereotactic microinjection system for transplanting cells. We evaluated the factors that affect cellular graft viability and other properties, including the gauge of the syringe needle and the injection rate. We systematically compared the synchronous withdrawal injection (SWI) and fixed-point injection (FPI) procedures in agarose and rat brain models.  assessments revealed superior dye dispersion with SWI compared to FPI, and  analyses confirmed that SWI reduced the tissue injury and improved cell distribution in the striatum. We applied this robot-assisted technique to evaluate the accuracy and safety of cell transplantation in canine models. Overall, this strategy enhances the accuracy and safety of graft delivery, potentially improving outcomes and advancing therapeutic strategies for the clinical treatment of neurodegenerative disorders.

Article	Year
Proteostasis of α-Synuclein and Its Role in the Pathogenesis of Parkinson's Disease. Frontiers in cellular neuroscience\| PubMed ID: 32210767	2020
Mesenchymal Stem/Stromal Cell-Mediated Mitochondrial Transfer and the Therapeutic Potential in Treatment of Neurological Diseases. Stem cells international\| PubMed ID: 32724315	2020
The Oxidative Damage and Inflammation Mechanisms in GERD-Induced Barrett's Esophagus. Frontiers in cell and developmental biology\| PubMed ID: 35721515	2022
Induced neural stem cells from Macaca fascicularis show potential of dopaminergic neuron specification and efficacy in a mouse Parkinson's disease model. Acta histochemica\| PubMed ID: 35792494	2022
Neuroprotective effects of human umbilical cord mesenchymal stromal cells in PD mice via centrally and peripherally suppressing NLRP3 inflammasome-mediated inflammatory responses. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 36076517	2022
Characterization of Human-Induced Neural Stem Cells and Derivatives following Transplantation into the Central Nervous System of a Nonhuman Primate and Rats. Stem cells international\| PubMed ID: 36618021	2022
Probiotic Formulation VSL#3 Interacts with Mesenchymal Stromal Cells To Protect Dopaminergic Neurons via Centrally and Peripherally Suppressing NOD-Like Receptor Protein 3 Inflammasome-Media... Microbiology spectrum\| PubMed ID: 36728426	2023
MicroRNAs of extracellular vesicles derived from mesenchymal stromal cells alleviate inflammation in dry eye disease by targeting the IRAK1/TAB2/NF-κB pathway. The ocular surface\| PubMed ID: 36990276	2023
Human iPSC-derived midbrain organoids functionally integrate into striatum circuits and restore motor function in a mouse model of Parkinson's disease. Theranostics\| PubMed ID: 37215566	2023
Modulation of human induced neural stem cell-derived dopaminergic neurons by DREADD reveals therapeutic effects on a mouse model of Parkinson's disease. Stem cell research & therapy\| PubMed ID: 39256801	2024
Robot-Assisted Stereotactic Microinjection Method for Precision Cell Transplantation in Rat and Canine Models. Cell transplantation\| PubMed ID: 40103182	2025

Deqiang Han

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