Stem Cell Research & Therapy

Mesenchymal stem cells (MSCs) are a promising source of stem cells for treating peripheral nerve injuries. Here, we present the first investigation of differentiation of olfactory mucosa-derived MSC (OM-MSC) towards a Schwann Cell (SC)-like phenotype. OM-MSCs are an advantageous potential source of SCs for peripheral nerve repair, as isolation can be accomplished with a minimally invasive procedure compared to autologous nerve harvest and isolation. Here, Schwann Cell Conditioned Media (SCCM) or a defined growth factor supplemented media (GF) was applied to OM-MSC for twenty-one days. The differentiation process and resulting populations were characterized by immunocytochemistry and RT-qPCR. Functionality of differentiated populations was assessed in an in vitro co-culture model to evaluate interaction with sensory neurons (dorsal root ganglia) juxtaposed to native SCs. Compared to undifferentiated MSCs, differentiation protocols resulted in significant changes in morphology, gene expression, and functionality using SCCM and GF media, representing key characteristics of SCs. Specifically, differentiated populations exhibit elongated, spindle-like morphologies, a high degree of eccentricity, increased S-100, CD44, and NGF expression, and colocalization of myelin basic proteins with neurites in the co-culture model. In conclusion, this work highlights the potential of OM-MSCs to be expanded and differentiated to SCs to improve synthetic scaffolds or for use in decellularized allografts for nerve repair.

BackgroundMesenchymal stem cells (MSCs) offer clinical promise for use in cell therapy approaches for regenerative medicine. A therapeutic challenge is that MSCs from different tissues are phenotypically and functionally distinct. Therefore, this study aims to molecularly characterize oral-derived MSCs by defining one of the three hallmarks of MSCs, differentiation potential, to discern their true molecular identities. MethodsThree different populations of oral tissue MSCs (from alveolar bone-aBMSCs; from dental pulp-DPSCs; and from gingiva-GMSCs) from three different patients were isolated and cultured. These MSCs were characterized for their stemness by flow cytometry and multi-differentiation potential, and their RNA was also isolated and analyzed quantitatively with RNA sequencing. Total mRNA-seq was performed and differentially expressed genes (DEGs) were identified in pairwise (DPSCs vs. aBMSCs, GMSCs vs. aBMSCs, and GMSCs vs. DPSCs) and tissue-specific comparisons (aBMSCs vs. Others, DPSCs vs. Others, GMSCs vs. Others) (FDR, p<0.05). Further, these DEGs, either common between MSC populations or unique to a specific MSC population, were evaluated for pathways and biological processes ResultsaBMSCs, DPSCs, and GMSCs were successfully isolated and characterized. The tissue-specific comparison revealed that DEGs were most numerous in DPSCs (693 genes) as compared to aBMSCs (103 genes) or DPSCs (232 genes). Statistically significant DEGs through pairwise comparisons present higher numbers in GMSCs vs. DPSCs (627) as compared to either DPSCs vs aBMSCs (286) or GMSCs vs. aBMSCs (82). Further analysis found that RUNX2, IBSP, SOX6, ACAN, and VCAM1 were significantly upregulated in aBMSCs. In DPSCs, BMP4 and IL6 were significantly downregulated, whereas AXL and NES were significantly upregulated. In GMSCs, AGPT1, SEMA4D, and PGDFA were significantly downregulated. Additionally, MAPK, PI3-AKT, and RAS signaling pathways were significantly regulated in GMSCs. Interestingly, aBMSCs and DPSCs revealed positive regulation of osteoblast differentiation, whereas GMSCs revealed negative regulation of osteoblast differentiation. DPSCs also revealed negative regulation of angiogenesis. ConclusionsOral-derived MSCs have an inherent "landscape" of differentiation defined by their tissue of origin; yet this differentiation potential can be modulated by their microenvironment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/606413v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@60aeceorg.highwire.dtl.DTLVardef@17480a2org.highwire.dtl.DTLVardef@1a9315aorg.highwire.dtl.DTLVardef@e74d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/468571v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12c091eorg.highwire.dtl.DTLVardef@ad1ccorg.highwire.dtl.DTLVardef@53ba90org.highwire.dtl.DTLVardef@fd65f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chemical reprogramming presents an innovative approach for generating induced pluripotent stem cells (iPSCs), bypassing the genetic instability and safe concern associated with viral vector approach. We describe a novel, efficient chemical method for reprogramming human umbilical cord tissue-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs). Compared to previous sources like adipose tissue and skin, frozen umbilical cord tissue offers an abundant, non-invasive, long-term storable, and ethically sound cell source. Our findings not only showcase the feasibility and safety of utilizing chemical reprogramming on cells from frozen umbilical cords but also underscore its potential in regenerative medicine, especially for developing safer and more effective therapies for cardiovascular diseases.

Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by: 1) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell amplification, 2) donor-specific characteristics including MSC frequency/quality that decline with disease state and increasing age, 3) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which pose safety and consistency issues. To overcome those limitations while enabling robust MSC production with constant high yield and quality, we developed a chemically defined biomimetic surface coating, called isoMATRIX, that facilitates the isolation of significantly higher numbers of MSCs in xeno-/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. In sum, the isoMATRIX promotes enhanced xeno-, serum-free, or chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate the structure and function of human fetal liver tissues, thus being considered as a promising tissue model for liver diseases and predictive compound screening. However, the adoption of HLOs in drug discovery faces several technical challenges, which include the lengthy differentiation process with multiple culture media leading to batch-to-batch variation, short-term maintenance of hepatic functions post-maturation, low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity levels compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. The dynamically cultured Diff-HLOs exhibited greater maturity and reproducibility than those cultured statically, especially after a 10-day differentiation period. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate liver recovery from DILI. The assessment of liver regeneration post-injury is critical to understanding the mechanism of recovery and determining the threshold drug concentration beyond which there will be a sharp decrease in the livers regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to the short-term differentiation of passage-able Exp-HLOs, stable hepatic function post-maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis. Graphical abstractThe overall process of dynamic liver organoid culture and in situ analysis in the 144PillarPlate/144PerfusionPlate for high-throughput hepatotoxicity assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/586638v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f2902aorg.highwire.dtl.DTLVardef@1d6e074org.highwire.dtl.DTLVardef@2a0dc0org.highwire.dtl.DTLVardef@eccde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human induced pluripotent stem cells (hiPSCs) hold considerable promise for autologous cellular therapies, particularly in ocular disease treatment, because of the low numbers of cells required for transplantation and the non-invasive nature of graft monitoring. However, the use of hiPSCs in ocular disease treatment faces challenges because the production of clinical-grade autologous hiPSCs via genetic techniques remains expensive, time-consuming, and subject to safety concerns. Here, we utilize a recently reported chemical method to derive human chemically induced pluripotent stem cells (hCiPSCs), demonstrating that hiPSC lines can be generated using small molecules in a simple and robust manner. Moreover, we show that these cell lines can be efficiently differentiated into retinal pigment epithelium (RPE) cells, which may aid in the treatment of age-related macular degeneration (AMD). Our study provides a foundation for cost-effective, fast, and safe methods that enable efficient production of autologous retinal cell types for ocular disease treatment.

Human fetal mesenchymal stem cells (hfMSCs) present advantageous characteristics compared to their adult counterparts and have emerged as potent cells in the field of regenerative medicine. In the context of skeletal regeneration, human amniotic fluid stem cells (AFSCs) have been shown to improve the quality and structure of the bone extracellular matrix in an experimental model of severe osteogenesis imperfecta. However, primary hfMSCs undergo replicative senescence during in vitro expansion, along with a progressive decrease in plasticity and tissue repair potential. To overcome this challenge, we rejuvenated AFSC to pluripotency using non-integrative episomal reprogramming and subsequently re-derived the cells towards the mesoderm to obtain induced MSCs (iMSCs). We found that iMSCs have a slower proliferation rate compared to their parental cell line (40h{+/-}2h vs. 29h{+/-}5h) but retain the multipotency and differentiation potential characteristic of MSCs. Comparative genomic analysis revealed that iMSCs express higher levels of genes involved in maintaining stemness, cell signaling, adhesion and migration, as well as promoting osteoblast differentiation, whilst AFSC expressed higher levels of genes involved in cell proliferation. In addition, iMSCs secrete small extracellular vesicles (iEVs) that have the potential to stimulate fibroblast migration, a key process in tissue repair and wound healing. Together, these data suggest that resetting the epigenetic clock of primary hfMSCs may represent a promising strategy to address the limitations associated with primary cell use and enhance their therapeutic potential.

Access to human neural cells is important for faithful in-vitro neural modeling as animals have innate species gaps. However, primary human cells are difficult to retrieve whereas immortalized human cell lines do not guarantee complete authenticity. Differentiation of human induced pluripotent stem cells (hiPSCs) has emerged as a novel approach to derive faithful human neural cells. Here we differentiated hiPSCs into functional midbrain dopaminergic (mDA) neurons and astrocytes by either a dual-SMAD method or a direct lineage reprogramming method. We found that a co-culture setting was necessary in maturing both the hiPSC-derived neurons and astrocytes. Besides, we showed that the neuron-astrocyte co-culture model was suitable for modeling the astrocyte-driven neuroinflammation. Overall, these hiPSC-derived neural cells represent powerful tools for both basic and translational studies.

IntroductionCyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM and UC upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility. MethodsMSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of three healthy female donors (BM-MSC-2-4), and from umbilical cords of two healthy male term infants (UC-MSC-1-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC and UC-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures. Given robust upregulation of CCL2 mRNA expression in HESCs exposed to the BM- and UC-MSC secretomes, transwell migration and invasion assays were performed to determine the effect of recombinant CCL2 on HESC motility. Statistical significance was defined as p<0.05. ResultsIndirect co-culture of HESCs with BM- or UC-MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. However, effects on cellular proliferation varied among the MSC donors. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,139 genes with FDR < 0.05. There was overlap among 4350 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2. Within four biological pathways enriched in HESCs, 4 genes (CCL2, HGF, PLAU, and BDKRB2) were differentially expressed in HESCs that had been cocultured with BM-MSC-1 and BM-MSC-2. qRT-PCR showed significantly increased mRNA expression of CCL2 in HESCs exposed to BM-MSC-1 (5-fold) and BM-MSC-2 (7.7-fold). In contrast, the increase in HGF expression was significant after exposure to BM-MSC-2 (1.8-fold) but not BM-MSC-1. Exposure to the UC-MSC secretome had similar effects on HESC-derived CCL2 and HGF levels. CCL2 expression was significantly increased (6.5-fold) by UC-MSC-2 but not by UC-MSC-1; HGF expression was significantly increased (1.6-fold) by UC-MSC-2 but not by UC-MSC-1. Validation studies indicated that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration (1.2-fold) and invasion (1.4-fold). These data suggest that CCL2 is a key factor in mediating MSC-induced HESC motility. ConclusionIncreased HESC motility by the secretome of BM- and UC-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by upregulated CCL2 expression in HESC. Together, our data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

Bovine embryonic stem cells (bESCs) serve as an optimal source for generating muscle and fat tissues. However, systematic methodologies for creating cultured beef from bESCs have not yet been reported. Here, we successfully established a bESC line and formulated a specialized culture medium that supports cell pluripotency in both two-dimensional (2D) and three-dimensional (3D) cultures. Additionally, we have also developed serum-free and transgene-free protocols that efficiently differentiate bESCs into muscle and fat cells. These differentiated cells were validated at the transcriptional and protein levels. Furthermore, we devised a method using high moisture stretched plant-based protein scaffolds for incorporating differentiated muscle and fat cells derived from bESCs, producing an innovative plant-cell hybrid cultured beef. These advancements provide a solid foundation for future cultured beef production using pluripotent stem cells.

At present, liver transplantation and hepatocyte therapy are common methods for the treatment of end-stage liver diseases, but they are restricted due to the shortage of liver donors and the safety and effectiveness of hepatocyte sources. Human adipose-derived mesenchymal stem cells (HAD-MSCs) have been applied to efficiently and stably induce phenotypic and functional liver cells or tissues in vitro due to their advantages such as wide sources and easy access to materials. In this study, the HAD-MSCs liver differentiation induction system was established and optimized based on the "cocktail method" of chemical small molecule compounds. We used HAD-MSCs as seed cells and gradually obtained mature hepatoid cells with normal phenotype and function after induction with small molecule compounds and growth factor system in vitro. The hepatoid cells induced by the two groups showed high similarity in phenotype and functional characteristics of mature hepatocytes. The differentiation system of human adipose mesenchymal stem cells into hepatocytes induced by small-molecule compounds in vitro was successfully constructed. This study will lay a foundation for the optimization of liver differentiation strategies and provide a reliable source of functional liver cells for clinical studies of liver diseases.

The columnar epithelial cells comprising the intestinal tract, stomach, and uterus can be cultured in vitro as organoids or in adherent culture. However, the proliferation of these columnar epithelial cells in adherent culture is limited. Likewise, human pluripotent stem cell (hPSC)-derived intestinal epithelial cells do not show extensive or clonal propagation in vitro. In this study, we induced proliferation of hPSC-derived small intestinal epithelium for a longer time by utilizing mesenchymal stromal cells derived from self-organized intestinal organoids as feeders. The proliferating cells exhibited columnar form, microvilli and glycocalyx formation, and cell polarity, as well as expression of drug-metabolizing enzymes and transporters. It is noteworthy that small intestinal epithelial stem cells cannot be cultured in adherent culture alone, and the stromal cells cannot be replaced by other feeders. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells, and play a crucial role in adherent culture. The high proliferative expansion, productivity, and functionality of hPSC-derived small intestinal epithelial stem cells could have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Very recent clinical advances in stem cell derived tissue replacement and gene therapy, in addition to the rise of artificial intelligence-aided scientific discovery, have placed the possibility of sophisticated human cell-based therapies firmly within reach. However, development of such cells and testing of their engineered gene circuit components, has proven highly challenging, due to the need for generating stable cell lines for each design-build-test-learn engineering cycle. Current approaches to generating stable human induced pluripotent stem cell (hiPSC) lines are highly time-consuming and suffer from lack of control, poor integration efficiency, and limited functionality. Validation in clinically relevant stem cell derived tissues is also broadly lacking. Such drawbacks are prohibitive to repeatably conducting cutting-edge stem cell engineering with broad application within a realistic timeframe and will not scale with the future of regenerative medicine. We have developed FAST-STEM (Facile Accelerated Stem-cell Transgene integration with SynBio Tunable Engineering Modes), a hPSC engineering platform that drastically reduces the time to generate differentiation ready stem cell lines from several weeks to 5 days, exhibiting a ~612-fold improvement in transgene integration rate over previous methodologies. Additional FAST-STEM innovations include: (i) rapid and highly efficient transgene integration; (ii) copy number control; (iii) simultaneous or consecutive integration of multiple gene cassettes; (iv) library screen capability. In addition to this unique functional versatility, platform transportability and broad use case for stem cell-engineering was confirmed by differentiation into eight different cell types across nine different laboratories. This platform dramatically lowers the bar for integration of synthetic biology with regenerative medicine, enabling experiments which were previously deemed logistically impossible, thus paving the way for sophisticated human cell device development.

Pancreatic islet transplantation is a promising treatment strategy for type 1 diabetes, however there are still major challenges to overcome, including vascularization. Novel strategies for the generation of prevascularized islets with native microvessels have reported improved islet functionality, vascularization and engraftment emphasizing integral role of microvascular bed. Recently a new model of self-organizing three-dimensional human blood vessel organoids (BVOs) has been developed from human pluripotent stem cells (hPSCs), composed of both endothelial and mural cells. BVO recapitulate key features of human microvasculature such as formation of vascular network, vascular lumen and basement membrane, and have been shown to be perfusable. Here, we report a new strategy to construct prevascularized islets by fusion with hPSC-derived BVOs. We demonstrate that islets and BVOs in co-culture leads to fusion and improved insulin secretion over time, on two independent human islet donors, suggesting a new therapeutic approach for pancreatic islet transplantation and type 1 diabetes modeling.

Infertility is thought to be caused by genetic mutations and dysfunction in the cellular niche where spermatogenesis takes place. An understanding of the specialized cellular processes which drive spermatogenesis is needed to develop treatments; however, the development of in vitro systems to study these cells has been hindered by our reliance on rarely available human testicular tissues for research. Human induced pluripotent stem cells (hiPSCs) can be used to derive human testicular-like cells, and thus provide an avenue for the development of in vitro testicular model systems. Therefore, this study set out to engineer a human testicular tissue model using hiPSCs for the first time. We demonstrate the ability of hiPSC-derived testicular cells to self-organize and mature into testicular-like tissues using organoid culture. Moreover, we show that hiPSC-derived testicular organoids promote testicular somatic cell maturation and spermatogenesis up to the post-meiotic spermatid stage. These hiPSC-derived testicular organoids have the potential to replace rarely available primary testicular tissues to further infertility research in an in vitro setting.

Spiral ganglion neurons (SGN) carry auditory information from sensory hair cells (HCs) to the brain. These auditory neurons, which are the target neurons of cochlear implants, degenerate following sensorineural hearing loss (SNHL). Prosthetic devices such as cochlear implants function by bypassing lost HCs and stimulating the residual SGNs, allowing restoration of hearing in deaf patients. Emerging cell-replacement therapies for SNHL include replacing damaged SGNs using stem cell-derived otic neuronal progenitors (ONPs). However, the availability of renewable, accessible, and patient-matched sources of human stem cells constitutes a major prerequisite towards cell replacement for auditory nerve recovery. Human dental pulp stem cells (hDPSCs) extracted from human wisdom teeth are self-renewing stem cells that originate from the neural crest during development. In this study, we developed a stepwise in vitro guidance procedure to differentiate hDPSCs into ONPs and then to SGNs. The procedure relies on the modulation of BMP and TGF-{beta} pathways for neurosphere formation as a first step, then a differentiation step based on two culture paradigms exploiting major signaling pathways (Wnt, Shh, RA) and neurotrophic factors involved in early otic neurogenesis. Gene and protein expression analyses revealed efficient induction of a comprehensive panel of known ONP and SGN-like cell markers over the course of in vitro differentiation. The use of atomic force microscopy revealed that hDPSC-derived SGN-like cells exhibit similar nanomechanical properties compared to their in vivo SGN counterparts. Furthermore, neurites extended between hDPSC-derived ONPs and rat SGN explants 4-6 days after co-culturing, suggesting the formation of neuronal contacts. These data indicate that the in vitro differentiated cells closely replicate the phenotypic and nanomechanical characteristics of human SGNs, advancing our culture differentiation system to the level to be used in next-generation cochlear implants and/or inner ear cell-based strategies for SNHL.

Human induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic, universal cells that could be transplanted to any recipient without requiring a matching donor, could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells, we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However, loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack, limiting graft survival. To counter this negative effect, we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF), an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression, combined with concurrent B2M and CIITA deletion, generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders.

In vitro maturation (IVM) is an infertility treatment used during in vitro fertilization (IVF) procedures in which immature oocytes are matured outside the body, limiting the excessive hormone doses required for retrieval of ready-to-fertilize oocytes. To overcome the historically low embryo formation rate associated with IVM, we have recently demonstrated that co-culture of hiPSC-derived ovarian support cells (OSCs) yielded higher rates of oocyte maturation and euploid embryo formation, by mimicking the complex ovarian environment in vitro, offering a novel solution to overcome the IVM main limitation. To translate this process into clinics, we sourced and engineered a compliant female clinical-grade (CG) hiPSC line to derive OSCs with similar quality attributes and clinical outcomes to results previously demonstrated with a research hiPSC line. We further optimized our manufacturing protocols to enable increased scale and substituted reagents with appropriate higher-quality alternatives. This strategic approach to product development has successfully met scalable manufacturing needs and ultimately resulted in a product of improved reproducibility, purity, and efficacy. Our findings support the use of a similar strategy to fine-tune hiPSC-derived products facilitating translation to clinical applications.