•Cell-free therapy using MSC-CM can offer an exciting approach in regenerative medicine.•Therapeutic role of cytokine and growth factors present in MSC-CM in clinical studies.•Cell-free therapy ...offers a significant advantage over cells based therapy and other conventional pharmaceutics.
Mesenchymal Stem Cells (MSCs) have been shown to be a promising candidate for cell-based therapy. The therapeutic potential of MSCs, towards tissue repair and wound healing is essentially based on their paracrine effects. Numerous pre-clinical and clinical studies of MSCs have yielded encouraging results. Further, these cells have been shown to be relatively safe for clinical applications. MSCs harvested from numerous anatomical locations including the bone marrow, adipose tissue, Wharton’s jelly of the umbilical cord etc., display similar immunophenotypic profiles. However, there is a large body of evidence showing that MSCs secrete a variety of biologically active molecules such as growth factors, chemokines, and cytokines. Despite the similarity in their immunophenotype, the secretome of MSCs appears to vary significantly, depending on the age of the host and niches where the cells reside. Thus, by implication, proteomics-based profiling suggests that the therapeutic potential of the different MSC populations must also be different. Analysis of the secretome points to its influence on varied biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic and anti-tumour for tissue maintenance and regeneration. Though MSC based therapy has been shown to be relatively safe, from a clinical standpoint, the use of cell-free infusions can altogether circumvent the administration of viable cells for therapy. Understanding the secretome of in vitro cultured MSC populations, by the analysis of the corresponding conditioned medium, will enable us to evaluate its utility as a new therapeutic option. This review will focus on the accumulating evidence that points to the therapeutic potential of the conditioned medium, both from pre-clinical and clinical studies. Finally, this review will emphasize the importance of profiling the conditioned medium for assessing its potential for cell-free therapy therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (
) account ...for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in
as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with
-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased
mRNA expression (quantitative real-time PCR qRT-PCR and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target
, an upstream regulator of
, to alter
expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that
expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.
Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of ...cells in the range of 0.5-2.5 × 10
cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Inherited retinal degenerations (IRD) encompasses a group of heterogeneous disorders causing debilitating visual diseases and blindness, affecting more than two million people worldwide, in all age ...groups. The inheritance patterns vary from autosomal dominant, autosomal recessive, X-linked, and sporadic with mutations in over 260 genes identified to date. Despite the significant advances in clinical diagnosis, there is no effective treatment available. Human-induced pluripotent stem cells (hiPSC) derived in vitro 3D retinal organoids offer a powerful preclinical tool to investigate the molecular mechanism(s) of inherited diseases. Organoids have the potential for the development of personalized therapies by modeling the disease-specific and patient-specific IRD. This mini-review will elaborate on the utility of the advanced culture model system by focusing on staging the in vitro human retinogenesis, modeling retinal diseases, and as a tool for testing potential therapeutic approaches to restore or prevent vision loss in affected individuals.
Purpose
There is a growing interest in extracellular vesicles (EVs) for ocular applications as therapeutics, biomarkers, and drug delivery vehicles. EVs secreted from mesenchymal stem cells (MSCs) ...have shown to provide therapeutic benefits in ocular conditions. However, very little is known about the properties of bioreactor cultured-3D human retinal organoids secreted EVs. This study provides a comprehensive morphological, nanomechanical, molecular, and proteomic characterization of retinal organoid EVs and compares it with human umbilical cord (hUC) MSCs.
Methods
The morphology and nanomechanical properties of retinal organoid EVs were assessed using Nanoparticle tracking analysis (NTA) and Atomic force microscopy (AFM). Gene expression analysis of exosome biogenesis of early and late retinal organoids were compared using qPCR. The protein profile of the EVs were analyzed with proteomic tools.
Results
NTA indicated the average size of EV as 100–250 nm. A high expression of exosome biogenesis genes was observed in late retinal organoids EVs. Immunoblot analysis showed highly expressed exosomal markers in late retinal organoids EVs compared to early retinal organoids EVs. Protein profiling of retinal organoid EVs displayed a higher differential expression of retinal function-related proteins and EV biogenesis proteins than hUCMSC EVs, implicating that the use of retinal organoid EVs may have a superior therapeutic effect on retinal disorders.
Conclusion
This study provides supplementary knowledge on the properties of retinal organoid EVs and suggests their potential use in the diagnostic and therapeutic treatments for ocular diseases.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
PurposeTransplanting photoreceptors from human pluripotent stem cell-derived retinal organoids have the potential to reverse vision loss in affected individuals. However, transplantable ...photoreceptors are only a subset of all cells in the organoids. Hence, the goal of our current study was to accelerate and synchronize photoreceptor differentiation in retinal organoids by inhibiting the Notch signaling pathway at different developmental time-points using a small molecule, PF-03084014 (PF). MethodsHuman induced pluripotent stem cell- and human embryonic stem cells-derived retinal organoids were treated with 10 µM PF for 3 days starting at day 45 (D45), D60, D90, and D120 of differentiation. Organoids were collected at post-treatment days 14, 28, and 42 and analyzed for progenitor and photoreceptor markers and Notch pathway inhibition by immunohistochemistry (IHC), quantitative PCR, and bulk RNA sequencing (n = 3-5 organoids from three independent experiments). ResultsRetinal organoids collected after treatment showed a decrease in progenitor markers (KI67, VSX2, PAX6, and LHX2) and an increase in differentiated pan-photoreceptor markers (OTX2, CRX, and RCVRN) at all organoid stages except D120. PF-treated organoids at D45 and D60 exhibited an increase in cone photoreceptor markers (RXRG and ARR3). PF treatment at D90 revealed an increase in cone and rod photoreceptors markers (ARR3, NRL, and NR2E3). Bulk RNA sequencing analysis mirrored the immunohistochemistry data and quantitative PCR confirmed Notch effector inhibition. ConclusionsTiming the Notch pathway inhibition in human retinal organoids to align with progenitor competency stages can yield an enriched population of early cone or rod photoreceptors.
Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene ( RHO ) ...account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO -CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR qRT-PCR and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3 , an upstream regulator of RHO , to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.
Advances in cancer nanotechnology had led to the development of various theranostics (therapy and diagnosis) strategies, by incorporating multifunctional approaches for trafficking against deadly ...cancer disease. In metastatic relapsed cancers, cancer stem cells (CSCs) exhibits drug resistance towards several therapies leading to their increased self-renewal, proliferation and differentiation. Hence, designing an effective strategy by employing the use of smart nanotheranostics can eliminate CSCs with the ultimate goal of enhancing the survival of cancer patients and offering a quality life. This review compiles the recent nanotheranostics strategies that are being employed for diagnosis, imaging and therapy of CSCs for circumventing the chances of cancer recurrence.
Schematic representation of (A) Cancer recurrence due to CSCs existence and (B) Cancer management by the application of nanotheranostics for diagnosis, imaging, and therapy of cancer stem cell. Display omitted
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
How cone photoreceptors are formed during retinal development is only partially known. This is in part because we do not fully understand the gene regulatory network responsible for cone genesis. We ...reasoned that cis-regulatory elements (enhancers) active in nascent cones would be regulated by the same upstream network that controls cone formation. To dissect this network, we searched for enhancers active in developing cones. By electroporating enhancer-driven fluorescent reporter plasmids, we observed that a sequence within an intron of the cone-specific Pde6c gene acted as an enhancer in developing mouse cones. Similar fluorescent reporter plasmids were used to generate stable transgenic human induced pluripotent stem cells that were then grown into three-dimensional human retinal organoids. These organoids contained fluorescently labeled cones, demonstrating that the Pde6c enhancer was also active in human cones. We observed that enhancer activity was transient and labeled a minor population of developing rod photoreceptors in both mouse and human systems. This cone-enriched pattern argues that the Pde6c enhancer is activated in cells poised between rod and cone fates. Additionally, it suggests that the Pde6c enhancer is activated by the same regulatory network that selects or stabilizes cone fate choice. To further understand this regulatory network, we identified essential enhancer sequence regions through a series of mutagenesis experiments. This suggested that the Pde6c enhancer was regulated by transcription factor binding at five or more locations. Binding site predictions implicated transcription factor families known to control photoreceptor formation and families not previously associated with cone development. These results provide a framework for deciphering the gene regulatory network that controls cone genesis in both human and mouse systems. Our new transgenic human stem cell lines provide a tool for determining which cone developmental mechanisms are shared and distinct between mice and humans.
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•An intronic enhancer within Pde6c drives expression in developing cones.•Transgenic retinal organoid systems show that the enhancer is active in human cones.•The Pde6c enhancer is activated by cells deciding between rod and cone fates.•The enhancer likely has five or more essential transcription factor binding regions.•Moderate conservation suggests flexibility of enhancer function between species.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human Amniotic Membranes (hAM) are an indispensable source as the components of the tissue engineering triad: scaffolds, cells, and bioactive molecules. Native collagen mimetic hAM is advantageous ...for biocompatibility, low cost, and availability as a scaffolding biomaterial for regenerative medicine. Recently, hAM has been recast as a potential source of composite biomaterial by various preparation techniques that aim at retaining growth factors and secretome. The exploration of hAM scaffolds its topography, its constituting array of bioactive molecules, and modality of application remain nascent. The wound healing cascade requisites elimination of over accumulated and deleterious Reactive Oxygen Species (ROS) to accelerate tissue repair and regenerative processes. The study reports decellularized, dehydrated Amniotic Membrane (dAM) incorporated with embelin, a natural benzoquinone compound synthesized from Embelia ribes for neutralizing free radicals, while simultaneously accelerates wound healing. hAM processed from the placenta has been characterized for integrity by histology, bio-degradability, thermogravimetric analysis (TGA), and cytokines analysis determined the presence of growth factors vital for tissue regeneration. The spectroscopic analysis confirmed the synthesized embelin and demonstrated burst release (>80%) from the embelin incorporated dAM supported by mathematical modelling. Surface topography and roughness of embelin incorporated AM were examined by scanning electron microscopy and atomic force microscopy respectively. In addition to anti-oxidant activity, the presence of embelin has significantly improved the initial fibroblast cell adhesion and proliferation compared to plain dAM and TCPS. In brief, the collagen mimetic intact dAM retains growth factors bioactivity and the anti-oxidant embelin synergistically influences the fibroblast cells thereby aid rapid wound healing and tissue regeneration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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