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The capability of human pluripotent stem cells (hPSCs) to self-renew and differentiate into any cell type has greatly contributed to the advancement of biomedicine. Reporter lines derived from hPSCs have played a crucial role in elucidating the mechanisms underlying human development and diseases by acting as an alternative reporter system that cannot be used in living humans. To bring hPSCs closer to clinical application in transplantation, scientists have generated reporter lines for isolating the desired cell populations, as well as improving graft quality and treatment outcomes. This review presents an overview of the applications of hPSC reporter lines and the important variables in designing a reporter system, including options for gene delivery and editing tools, design of reporter constructs, and selection of reporter genes. It also provides insights into the prospects of hPSC reporter lines and the challenges that must be overcome to maximize the potential of hPSC reporter lines.
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Facial infiltrating lipomatosis (FIL) is a congenital disorder. The pathogenesis of FIL is associated with PIK3CA mutations, but the underlying mechanisms remain undetermined. We found that the adipose tissue in FIL demonstrated adipocytes hypertrophy and increased lipid accumulation. All adipose-derived mesenchymal stem cells from FIL (FIL-ADSCs) harbored PIK3CA mutations. Moreover, FIL-ADSCs exhibited a greater capacity for adipogenesis. Knockdown of PIK3CA resulted in a reduction in the adipogenic potential of FIL-ADSCs. Furthermore, WX390, a dual-target PI3K/mTOR inhibitor, was found to impede PIK3CA-mediated adipogenesis both in vivo and in vitro. RNA sequencing (RNA-seq) revealed that the expression of transient receptor potential vanilloid subtype 1 (TRPV1) was upregulated after PI3K pathway inhibition, and overexpression or activation of TRPV1 both inhibited adipogenesis. Our study showed that PIK3CA mutations promoted adipogenesis in FIL-ADSCs and this effect was achieved by suppressing TPRV1. Pathogenesis experiments suggested that WX390 may serve as an agent for the treatment of FIL.
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Lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs) relies on complex interactions between biochemical and physical cues. Here we investigated the ability of hiPSCs to undergo lineage commitment in response to inductive signals and assessed how this competence is modulated by substrate stiffness. We showed that Activin A-induced hiPSC differentiation into mesendoderm and its derivative, definitive endoderm, is enhanced on gel-based substrates softer than glass. This correlated with changes in tight junction formation and extensive cytoskeletal remodeling. Further, live imaging and biophysical studies suggested changes in cell motility and interfacial contacts underlie hiPSC layer reshaping on soft substrates. Finally, we repurposed an ultra-soft silicone gel, which may provide a suitable substrate for culturing hiPSCs at physiological stiffnesses. Our results provide mechanistic insight into how epithelial mechanics dictate the hiPSC response to chemical signals and provide a tool for their efficient differentiation in emerging stem cell therapies.
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In response to corneal injury, an activation of corneal epithelial stem cells and their direct progeny the early transit amplifying (eTA) cells to rapidly proliferate is critical for proper re-epithelialization. Thus, it is important to understand how such stem/eTA cell activation is regulated. Angiotensin-converting enzyme 2 (ACE2) is predominantly expressed in the stem/eTA-enriched limbal epithelium but its role in the limbal epithelium was unclear. Single cell RNA sequencing (scRNA-seq) suggested that Ace2 involved the proliferation of the stem/eTA cells. Ace2 was reduced following corneal injury. Such reduction enhanced limbal epithelial proliferation and downregulated LCN2, a negative regulator of proliferation in a variety of tissues, via upregulating TGFA and consequently activating epidermal growth factor receptor (EGFR). Inhibition of EGFR or overexpression of LCN2 reversed the increased proliferation in limbal epithelial cells lacking ACE2. Our findings demonstrate that after corneal injury, ACE2 is downregulated, which activates limbal epithelial cell proliferation via a TGFA/EGFR/LCN2 pathway.
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Neural diseases are challenging to treat and are regarded as one of the major causes of disability and morbidity in the world. Stem cells can provide a solution, by offering a mechanism to replace damaged circuitry. However, obtaining sufficient cell sources for neural regeneration remains a significant challenge. In recent years, waste-derived stem(-like) cells (WDS-lCs) extracted from both prenatal and adult clinical waste tissues/products, have gained increasing attention for application in neural tissue repair and remodeling. This often-overlooked pool of cells possesses favorable characteristics; including self-renewal, neural differentiation, secretion of neurogenic factors, cost-effectiveness, and low ethical concerns. Here, we offer a perspective regarding the biological properties, extraction protocols, and preclinical and clinical treatments where prenatal and adult WDS-lCs have been utilized for cell replacement therapy in neural applications, and the challenges involved in optimizing these approaches toward patient led therapies.
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Stem cell therapies for degenerative cartilage disease are limited by an incomplete understanding of hyaline cartilage formation and maintenance. Human bone marrow stromal cells/skeletal stem cells (hBMSCs/SSCs) produce stable hyaline cartilage when attached to hyaluronic acid-coated fibrin microbeads (HyA-FMBs), yet the mechanism remains unclear. In vitro, hBMSC/SSC/HyA-FMB organoids exhibited reduced BMP signaling early in chondrogenic differentiation, followed by restoration of BMP signaling in chondrogenic IGFBP5 + /MGP + cells. Subsequently, human-induced pluripotent stem cell (hiPSC)-derived sclerotome cells were established (BMP inhibition) and then treated with transforming growth factor ß (TGF-ß) -/+ BMP2 and growth differentiation factor 5 (GDF5) (BMP signaling activation). TGF-ß alone elicited a weak chondrogenic response, but TGF-ß/BMP2/GDF5 led to delamination of SOX9 + aggregates (chondrospheroids) with high expression of COL2A1, ACAN, and PRG4 and minimal expression of COL10A1 and ALP in vitro. While transplanted hBMSCs/SSCs/HyA-FMBs did not heal articular cartilage defects in immunocompromised rodents, chondrospheroid-derived cells/HyA-FMBs formed non-hypertrophic cartilage that persisted until at least 5 months in vivo.
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Adult stem cells play a critical role in tissue repair and maintenance. In tissues with slow turnover, including skeletal muscle, these cells are maintained in a mitotically quiescent state yet remain poised to re-enter the cell cycle to replenish themselves and regenerate the tissue. Using a panomics approach we show that the PAX7/NEDD4L axis acts against muscle stem cell activation in homeostatic skeletal muscle. Our findings suggest that PAX7 transcriptionally activates the E3 ubiquitin ligase Nedd4L and that the conditional genetic deletion of Nedd4L impairs muscle stem cell quiescence, with an upregulation of cell cycle and myogenic differentiation genes. Loss of Nedd4L in muscle stem cells results in the expression of doublecortin (DCX), which is exclusively expressed during their in vivo activation. Together, these data establish that the ubiquitin proteasome system, mediated by Nedd4L, is a key contributor to the muscle stem cell quiescent state in adult mice.
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Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and myeloid skewing. We report that culture of bone marrow (BM) HSCs from aged mice with epidermal growth factor (EGF) suppressed myeloid skewing, increased multipotent colony formation, and increased HSC repopulation in primary and secondary transplantation assays. Mice transplanted with aged, EGF-treated HSCs displayed increased donor cell engraftment within BM HSCs and systemic administration of EGF to aged mice increased HSC self-renewal capacity in primary and secondary transplantation assays. Expression of a dominant negative EGFR in Scl/Tal1+ hematopoietic cells caused increased myeloid skewing and depletion of long term-HSCs in 15-month-old mice. EGF treatment decreased DNA damage in aged HSCs and shifted the transcriptome of aged HSCs from genes regulating cell death to genes involved in HSC self-renewal and DNA repair but had no effect on HSC senescence. These data suggest that EGFR signaling regulates the repopulating capacity of aged HSCs.
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Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.
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Mutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.
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Stem cell therapy for intrauterine adhesions (IUAs) has been widely used in clinical treatment. However, intravenous injection lacks sufficient targeting capabilities, while in situ injection poses challenges in ensuring the effective survival of stem cells. Furthermore, the mechanism underlying the interaction between stem cells and endometrial cells in vivo remains poorly understood, and there is a lack of suitable in vitro models for studying these problems. Here, we designed an extracellular matrix (ECM)-adhesion mimic hydrogel for intrauterine administration, which was more effective than direct injection in treating IUAs. Additionally, we analyzed the epithelial-mesenchymal transition (EMT) and confirmed that the activation of endometrial epithelial stem cells is pivotal. Our findings demonstrated that umbilical cord mesenchymal stem cells (UC-MSCs) secrete WNT7A to activate endometrial epithelial stem cells, thereby accelerating regeneration of the endometrial epithelium. Concurrently, under transforming growth factor alpha (TGFA) stimulation secreted by the EMT epithelium, UC-MSCs upregulate E-cadherin while partially implanting into the endometrial epithelium.
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Inflammatory bowel disease (IBD) is a chronic disorder with an increasing global prevalence. Managing disease activity relies on various pharmacological options. However, the effectiveness of current therapeutics is limited and not universally applicable to all patients and circumstances. Consequently, developing new management strategies is necessary. Recent advances in endoscopically obtained intestinal biopsy specimens have highlighted the potential of intestinal epithelial organoid transplantation as a novel therapeutic approach. Experimental studies using murine and human organoid transplantations have shown promising outcomes, including tissue regeneration and functional recovery. Human trials with organoid therapy have commenced; thus, this article provides readers with insights into the necessity and potential of intestinal organoid transplantation as a new regenerative therapeutic option in clinical settings and explores its associated challenges.
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The intestine is vulnerable to chemotherapy-induced damage due to the high rate of intestinal epithelial cell (IEC) proliferation. We have developed a human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced IEC damage on T cell behavior. Exposure of intestinal organoids to busulfan, fludarabine, and clofarabine induced damage-related responses affecting both the capacity to regenerate and transcriptional reprogramming. In ex vivo co-culture assays, prior intestinal organoid damage resulted in increased T cell activation, proliferation, and migration. We identified galectin-9 (Gal-9) as a key molecule released by damaged organoids. The use of anti-Gal-9 blocking antibodies or CRISPR/Cas9-mediated Gal-9 knock-out prevented intestinal organoid damage-induced T cell proliferation, interferon-gamma release, and migration. Increased levels of Gal-9 were found early after HSCT chemotherapeutic conditioning in the plasma of patients who later developed acute GVHD. Taken together, chemotherapy-induced intestinal damage can influence T cell behavior in a Gal-9-dependent manner which may provide novel strategies for therapeutic intervention.
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Cancer-associated fibroblasts (CAFs) play a major role in reorganizing the physical tumor micro-environment and changing tissue stiffness. Herein, using an engineered three-dimensional (3D) model that mimics the tumor's native biomechanical environment, we characterized the changes in matrix stiffness caused by six patient-specific colorectal CAF populations. After 21 days of culture, atomic force microscopy (AFM) was performed to precisely measure the local changes in tissue stiffness. Each CAF population exhibited heterogeneity in remodeling capabilities, with some patient-derived cells stiffening the matrix and others softening it. Tissue stiffening was mainly attributed to active contraction of the matrix by the cells, whereas the softening was due to enzymatic activity of matrix-cleaving proteins. This measured heterogeneity was lost when the CAFs were cocultured with colorectal cancer cells, as all samples significantly soften the tissue. The interplay between cancer cells and CAFs was critical as it altered any heterogeneity exhibited by CAFs alone.
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Hypertension is a major cause of morbidity and mortality in patients with hypertrophic cardiomyopathy (HCM), suggesting a potential role for mechanics in HCM pathogenesis. Here, we developed an in vitro physiological model to investigate how mechanics acts together with HCM-linked myosin binding protein C (MYBPC3) mutations to trigger disease. Micro-heart muscles (µHM) were engineered from induced pluripotent stem cell (iPSC)-derived cardiomyocytes bearing MYBPC3+/- mutations and challenged to contract against substrates of different elasticity. µHMs that worked against substrates with stiffness at or exceeding the stiffness of healthy adult heart muscle exhibited several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in MYBPC3+/- µHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca2+ intake through membrane-embedded channels underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease with iPSC technology.
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The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aims to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPs) and pancreas progenitors (PPs), without using Matrigel. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP + MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP + MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development.
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Allergic asthma is a chronic non-communicable disease characterized by lung tissue inflammation. Current treatments can alleviate the clinical symptoms to some extent, but there is still no cure. Recently, the transplantation of mesenchymal stem cells (MSCs) has emerged as a potential approach for treating allergic asthma. Gingival-derived mesenchymal stem cells (GMSCs), a type of MSC recently studied, have shown significant therapeutic effects in various experimental models of autoimmune diseases. However, their application in allergic diseases has yet to be fully elucidated. In this study, using an OVA-induced allergic asthma model, we demonstrated that GMSCs decrease CD11b+CD11c+ proinflammatory dendritic cells (DCs), reduce Th2 cells differentiation, and thus effectively diminish eosinophils infiltration. We also identified that the core functional factor, hepatocyte growth factor (HGF) secreted by GMSCs, mediated its effects in relieving airway inflammation. Taken together, our findings indicate GMSCs as a potential therapy for allergic asthma and other related diseases.
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Previous studies have indicated the neuroprotective effect of olfactory mucosa mesenchymal stem cells (OM-MSCs) on brain injury. Intracerebral hemorrhage (ICH) models were established in rats by injecting autologous blood. SENP1 expression was enhanced in neurons but decreased in astrocytes compared to that in OM-MSCs. Overexpression of SENP1 promoted the proliferation and neuronal differentiation, while inhibiting the astrocytic differentiation of OM-MSCs. Conversely, its knockdown had the opposite effect. Moreover, OM-MSCs reduced neurological dysfunction in rats after ICH, and the neuroprotective effect of OM-MSCs could be further enhanced by SENP1 overexpression. In addition, SENP1 promoted mitophagy, which might be related to SENP1-mediated OPTN deSUMOylation. Furthermore, SENP1 promoted neuronal differentiation of OM-MSCs through mitophagy mediated by OPTN. Similar to SENP1, OPTN transfection further enhanced the remission effect of OM-MSC on ICH rats. SENP1 promoted neuronal differentiation of OM-MSCs through OPTN-mediated mitophagy to improve neurological deficits in ICH rats.
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Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial, we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally, both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent.
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Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been proposed as a treatment for graft-versus-host disease (GVHD), which is a major complication following allogeneic hematopoietic cell transplantation. However, clinical trials have not yielded good results, and human decidua-derived mesenchymal stromal cells (DSCs) have been proposed as an alternative. In addition, the mechanism by which DSCs exert their immunomodulatory effects is still unknown. We found that knockdown of IL-6 in DSCs reduced the expression of PD-L1 and PD-L2, which are known as classical immune checkpoint inhibitors. Expression of PD-L1 and PD-L2 was restored by adding recombinant IL-6 to the DSCs. When DSCs and IL-6-knockdown DSCs were administered as treatment in a murine GVHD model, the group receiving IL-6-knockdown DSCs had significantly higher mortality and clinical scores compared to the group receiving DSCs. Taken together, these data suggest that the IL-6 signaling pathway is a crucial contributor to the immunosuppressive capacity of DSCs.