Stem Cell Fate Specification Research Articles

Pathway to Independence - an interview with Clémentine Villeneuve.

Clémentine Villeneuve is a Postdoctoral Researcher at the Max Planck Institute for Molecular Biomedicine, Germany, where her research focuses on the role of mechanobiology in driving stem cell fate specification and tissue patterning during development. Clémentine is one of the fellows of Development's Pathway to Independence Programme, and we caught up with her over Zoom to find out more about her research background, her hopes for the programme and her future research questions.

Enhancing Bone Regeneration through CDC20-Loaded ZIF-8 Nanoparticles Wrapped in Erythrocyte Membranes with Targeting Aptamer.

In the context of bone regeneration, nanoparticles harboring osteogenic factors have emerged as pivotal agents for modulating the differentiation fate of stem cells. However, persistent challenges surrounding biocompatibility, loading efficiency, and precise targeting ability warrant innovative solution. In this study, a novel nanoparticle platform founded upon the zeolitic imidazolate framework-8 (ZIF-8) is introduced. This new design, CDC20@ZIF-8@eM-Apt, involves the envelopment of ZIF-8 within an erythrocyte membrane (eM) cloak, and is coupled with a targeting aptamer. ZIF-8, distinguished by its porosity, biocompatibility, and robust cargo transport capabilities, constitutes the core framework. Cell division cycle protein 20 homolog (CDC20) is illuminated as a new target in bone regeneration. The eM plays a dual role in maintaining nanoparticle stability and facilitating fusion with target cell membranes, while the aptamer orchestrates the specific recruitment of bone marrow mesenchymal stem cells (BMSCs) within bone defect sites. Significantly, CDC20@ZIF-8@eM-Apt amplifies osteogenic differentiation of BMSCs via the inhibition of NF-κB p65, and concurrently catalyzes bone regeneration in two bone defect models. Consequently, CDC20@ZIF-8@eM-Apt introduces a pioneering strategy for tackling bone defects and associated maladies, opening novel avenues in therapeutic intervention.

Manipulation of Stem Cell Fate In Vitro Using Plasmid-Based Transcription Factor Over-Expression Systems

Introduction: The ability to switch stem cell differentiation fate in-vitro, is a powerful tool that may allow the generation of large numbers of cells that may be required in order to develop biologically engineered tissues and cells required for therapeutic applications such as pharmacological testing of new medications. The transcription factor (“master switch”) Olig2, alone or in conjunction with Nkx2.2, has been implicated as a key cell fate decider for emerging neuro-glial precursors derived from both embryonic stem (ES) cells and from foetal neural stem (FNS) cells. Methods: The in-vitro system of stem cells devoid of exogenous signaling was developed. Stem cells were manipulated by pIRES plasmid vector driven, constitutively expressed Olig-2 or Olig-2/Nkx2.2 transcription factor system introduced into proliferating embryonic or foetal neural stem cells, following a similar embryological temporal p at te r ni n g s e q ue nc e s e e n i n -vi v o. Findings: Successful stem cell fate modification could be achieved in-vitro using the transcription factor overexpression system. Substantially different cell fates were noted in the presence of Olig-2 alone and in combination with Nkx2.2, with the achievement of premature glial differentiation. Conclusion: This method, therefore, may be useful to generate rare live human cells (such as Oligodendroglia or specialised myocardial cells) in-vitro.

Ginsenosides on stem cells fate specification-a novel perspective.

Recent studies have demonstrated that stem cells have attracted much attention due to their special abilities of proliferation, differentiation and self-renewal, and are of great significance in regenerative medicine and anti-aging research. Hence, finding natural medicines that intervene the fate specification of stem cells has become a priority. Ginsenosides, the key components of natural botanical ginseng, have been extensively studied for versatile effects, such as regulating stem cells function and resisting aging. This review aims to summarize recent progression regarding the impact of ginsenosides on the behavior of adult stem cells, particularly from the perspective of proliferation, differentiation and self-renewal.

Engineered Living Oriented Electrospun Fibers Regulate Stem Cell Para‐Secretion and Differentiation to Promote Spinal Cord Repair (Adv. Healthcare Mater. 9/2023)

Electrospinning In article 2202785 by Liang Chen, Wenguo Cui, Yong Gu, and co-workers, a living-oriented fiber scaffold is found to fully exploit the advantage of stem cells, which can respond not only to regulate the local inflammatory microenvironment at the early stages of spinal cord injury, but also to further regulate the differentiation fate of stem cells, continuously release brain-derived neurotrophic factor, and promote neuronal regeneration.

Piezo protein determines stem cell fate by transmitting mechanical signals.

Piezo ion channel is a mechanosensitive protein on the cell membrane, which contains Piezo1 and Piezo2. Piezo channels are activated by mechanical forces, including stretch, matrix stiffness, static pressure, and shear stress. Piezo channels transmit mechanical signals that cause different downstream responses in the differentiation process, including integrin signaling pathway, ERK1/2 MAPK signaling pathway, Notch signaling, and WNT signaling pathway. In the fate of stem cell differentiation, scientists found differences in Piezo channel expression and found that Piezo channel expression is related to developmental diseases. Here, we briefly review the structure and function of Piezo channels and the relationship between Piezo and mechanical signals, discussing the current understanding of the role of Piezo channels in stem cell fate and associated molecules and developmental diseases. Ultimately, we believe this review will help identify the association between Piezo channels and stem cell fate.

Paxillin tunes the relationship between cell-matrix and cell-cell adhesions to regulate stiffness-dependent dentinogenesis.

Mechanical stiffness is recognized as a key physical factor and directs cell function via a mechanotransduction process, from extracellular physical cues to intracellular signaling cascades that affect transcriptional activity. Cells continually receive mechanical signals from both the surrounding matrix and adjacent cells. However, how mechanical stiffness cue at cell-substrate interfaces coordinates cell-cell junctions in guiding mesenchymal stem cell behaviors is poorly understood. Here, polydimethylsiloxane substrates with different stiffnesses were used to study mechanosensation/transduction mechanisms in controlling odontogenic differentiation of dental papilla cells (DPCs). DPC phenotypes (morphology and differentiation) changed in response to the applied force derived from stiff substrates. Significantly, higher expression of paxillin on stiffer substrates promoted DPC dentinogenesis. Upon treatment with siRNA to knockdown paxillin, N-cadherin increased mainly in the cytomembrane at the area of cell-cell contacts, whereas β-catenin decreased in the nuclei. The result of a double luciferase reporter assay showed that stiffness promoted β-catenin binding to TCF, which could coactivate the target genes associated with odontogenic differentiation, as evidenced by bioinformatics analysis. Finally, we determined that the addition of a β-catenin inhibitor suppressed DPC mineralization in all the stiffness groups. Thus, our results indicated that a mechanotransduction process from cell-substrate interactions to cell-cell adhesions was required for DPC odontogenic differentiation under the stimulation of substrate stiffness. This finding suggests that stem cell fate specification under the stimulus of stiffness at the substrates is based on crosstalk between substrate interactions and adherens junctions, which provides an essential mechanism for cell-based tissue engineering.

Tracing the developmental origin of tissue stem cells.

Tissue stem cells are vital for organ homeostasis and regeneration owing to their ability to self-renew and differentiate into the various cell types that constitute organ tissue. These stem cells are formed during complex and dynamic organ development, necessitating spatial-temporal coordination of morphogenetic events and cell fate specification during this process. In recent years, technological advances have enabled the tracing of the cellular dynamics, states, and lineages of individual cells over time in relation to tissue morphological changes. These dynamic data have not only revealed the origin of tissue stem cells in various organs but have also led to an understanding of the molecular, cellular, and biophysical bases of tissue stem cell formation. Herein, we summarize recent findings on the developmental origin of tissue stem cells in the hair follicles, intestines, brain, skeletal muscles, and hematopoietic system, and further discuss how stem cell fate specification is coordinated with tissue topology.

Carbon Quantum Dots for Stem Cell Imaging and Deciding the Fate of Stem Cell Differentiation

Nanotechnology advancements and applications have paved the way for new possibilities in regenerative medicine and tissue engineering. It is a relatively new field that has the potential to improve stem cell differentiation and therapy greatly. Numerous studies have demonstrated that nanomaterials can function as a physiological niche for the formation and differentiation of stem cells. However, quantum dots (QDs), such as carbon quantum dots (CQDs) and graphene quantum dots (GQDs), have shown considerable promise in the field of regenerative medicine. To date, most research has focused on stem cell tracking and imaging using CQDs. However, their interaction with stem cells and the associated possibility for differentiation by selectively focusing chemical signals to a particular lineage has received scant attention. In this mini-review, we attempt to categorize a few pathways linked with the role of CQDs in stem cell differentiation.

Microenvironmentally optimized 3D-printed TGFβ-functionalized scaffolds facilitate endogenous cartilage regeneration in sheep

Clinically, microfracture is the most commonly applied surgical technique for cartilage defects. However, an increasing number of studies have shown that the clinical improvement remains questionable, and the reason remains unclear. Notably, recent discoveries revealed that signals from regenerated niches play a critical role in determining mesenchymal stem cell fate specification and differentiation. We speculate that a microenvironmentally optimized scaffold that directs mesenchymal stem cell fate will be a good therapeutic strategy for cartilage repair. Therefore, we first explored the deficiency of microfractures in cartilage repair. The microfracture not only induced inflammatory cell aggregation in blood clots but also consisted of loose granulation tissue with increased levels of proteins related to fibrogenesis. We then fabricated a functional cartilage scaffold using two strong bioactive cues, transforming growth factor-β3 and decellularized cartilage extracellular matrix, to modulate the cell fate of mesenchymal stem cells. Additionally, poly(ε-caprolactone) was also coprinted with extracellular matrix-based bioinks to provide early mechanical support. The in vitro studies showed that microenvironmentally optimized scaffolds exert powerful effects on modulating the mesenchymal stem cell fate, such as promoting cell migration, proliferation and chondrogenesis. Importantly, this strategy achieved superior regeneration in sheep via scaffolds with biomechanics (restored well-organized collagen orientation) and antiapoptotic properties (cell death-related genes were also downregulated). In summary, this study provides evidence that microenvironmentally optimized scaffolds improve cartilage regeneration in situ by regulating the microenvironment and support further translation in human cartilage repair. Statement of significanceAlthough microfracture (MF)-based treatment for chondral defects has been commonly used, critical gaps exist in understanding the biochemistry of MF-induced repaired tissue. More importantly, the clinically unsatisfactory effects of MF treatment have prompted researchers to focus on tissue engineering scaffolds that may have sufficient therapeutic efficacy. In this manuscript, a 3D printing ink containing cartilage tissue-specific extracellular matrix (ECM), methacrylate gelatin (GelMA), and transforming growth factor-β3 (TGF-β3)-embedded polylactic-coglycolic acid (PLGA) microspheres was coprinted with poly(ε-caprolactone) (PCL) to fabricate tissue engineering scaffolds for chondral defect repair. The sustained release of TGF-β3 from scaffolds successfully directed endogenous stem/progenitor cell migration and differentiation. This microenvironmentally optimized scaffold produced improved tissue repair outcomes in the sheep animal model, explicitly guiding more organized neotissue formation and therefore recapitulating the anisotropic structure of native articular cartilage. We hypothesized that the cell-free scaffolds might improve the clinical applicability and become a new therapeutic option for chondral defect repair.

Transcriptome Profile of Membrane and Extracellular Matrix Components in Ligament-Fibroblastic Progenitors and Cementoblasts Differentiated from Human Periodontal Ligament Cells

Ligament-fibroblastic cells and cementoblasts, two types of progenitor cells that differentiate from periodontal ligament stem cells (hPDLSCs), are responsible for the formation of the adhesive tissues in the tooth root. Since one of the factors that determines the fate of stem cell differentiation is the change in the microenvironment of the stem/progenitor cells, this study attempted to compare and analyze the molecular differences in the membrane and ECM of the two progenitor cells. Single cells derived from hPDLSCs were treated with TGF-β1 and BMP7 to obtain ligament-fibroblastic and cementoblastic cells, respectively. The transcriptome profiles of three independent replicates of each progenitor were evaluated using next-generation sequencing. The representative differentially expressed genes (DEGs) were verified by qRT-PCR, Western blot analysis, and immunohistochemistry. Among a total of 2245 DEGs identified, 142 and 114 DEGs related to ECM and cell membrane molecules were upregulated in ligament-fibroblastic and cementoblast-like cells, respectively. The major types of integrin and cadherin were found to be different between the two progenitor cells. In addition, the representative core proteins for each glycosaminoglycan-specific proteoglycan class were different between the two progenitors. This study provides a detailed understanding of cell–cell and cell–ECM interactions through the specific components of the membrane and ECM for ligament-fibroblastic and cementoblastic differentiation of hPDLSCs.

Injectable, viscoelastic hydrogel precisely regulates developmental tissue regeneration

Extracellular matrix viscoelasticity is a crucial parameter for designing biomaterials in tissue regeneration. However, the precise control of viscoelastic properties for reproducing the mechanical environment that cells experience in vivo remains a challenge. Here, we developed a series of injectable, viscoelastic PEGylated poly (glycerol sebacate) (PEGS-OH) hydrogels crosslinked via click chemistry and investigated the role of viscoelasticity in developmental tissue regeneration. We found that, compared to 100% and 60% crosslink degrees, 80% crosslinked PEGS-OH with incomplete network slipping promoted a striking increase in the adhesion and differentiation of bone mesenchymal stem cells (BMSCs). Moreover, with PEG segments increasing, the elastic hydrogel turned into viscous state and regulated BMSCs differentiation from osteogenesis to adipogenesis. Further studies revealed that the crosslink degree- and PEG segment-induced BMSCs response was greatly relied on the mechanosensitive Piezo1 mechanism. With in vivo cartilago articularis and skull defect regeneration as models, tendencies from experiments in vitro were further confirmed. Our findings highlight a potential material-biological strategy to precisely regulate cell fate and ensuing tissue regeneration, and provide insight into the relevance of material viscoelasticity in the fate of stem cell differentiation.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

MeCP2 controls neural stem cell fate specification through miR-199a-mediated inhibition of BMP-Smad signaling.

Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

Macropore design of tissue engineering scaffolds regulates mesenchymal stem cell differentiation fate

Craniosynostosis is a debilitating birth defect characterized by the premature fusion of cranial bones resulting from premature loss of stem cells located in suture tissue between growing bones. Mesenchymal stromal cells in long bone and the cranial suture are known to be multipotent cell sources in the appendicular skeleton and cranium, respectively. We are developing biomaterial constructs to maintain stemness of the cranial suture cell population towards an ultimate goal of diminishing craniosynostosis patient morbidity. Recent evidence suggests that physical features of synthetic tissue engineering scaffolds modulate cell and tissue fate. In this study, macroporous tissue engineering scaffolds with well-controlled spherical pores were fabricated by a sugar porogen template method. Cell-scaffold constructs were implanted subcutaneously in mice for up to eight weeks then assayed for mineralization, vascularization, extracellular matrix composition, and gene expression. Pore size differentially regulates cell fate, where sufficiently large pores provide an osteogenic niche adequate for bone formation, while sufficiently small pores (<125 μm in diameter) maintain stemness and prevent differentiation. Cell-scaffold constructs cultured in vitro followed the same pore size-controlled differentiation fate. We therefore attribute the differential cell and tissue fate to scaffold pore geometry. Scaffold pore size regulates mesenchymal cell fate, providing a novel design motif to control tissue regenerative processes and develop mesenchymal stem cell niches in vivo and in vitro through biophysical features.

A bioenergetic shift is required for spermatogonial differentiation

A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference for bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid (RA)-induced differentiation. To accommodate this elevated energy demand, RA signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. Disrupting mitochondrial respiration significantly blocked spermatogonial differentiation. Inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway also repressed the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon RA-induced differentiation, with both RNA-seq and quantitative proteomic analyses. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration that is required for spermatogonial proliferation and differentiation.

The Effect of Curcumin on the Differentiation of Mesenchymal Stem Cells into Mesodermal Lineage.

Curcumin has been placed at the forefront of the researcher’s attention due to its pleiotropic pharmacological effects and health benefits. A considerable volume of articles has pointed out curcumin’s effects on the fate of stem cell differentiation. In this review, a descriptive mechanism of how curcumin affects the outcome of the differentiation of mesenchymal stem cells (MSCs) into the mesodermal lineage—i.e., adipocyte, osteocyte, and chondrocyte differentiation—is compiled from the literature. The sections include the mechanism of inhibition or induction of MSCs differentiation to each lineage, their governing molecular mechanisms, and their signal transduction pathways. The effect of different curcumin doses and its structural modifications on the MSCs differentiation is also discussed.

Chirality Controls Mesenchymal Stem Cell Lineage Diversification through Mechanoresponses

Biogenesis and tissue development are based on the heterogenesis of multipotent stem cells. However, the underlying mechanisms of stem cell fate specification are unclear. Chirality is one of the most crucial factors that affects stem cell development and is implicated in asymmetrical cell morphology formation; however, its function in heterogeneous cell fate determination remains elusive. In this study, it is reported that the chirality of a constructed 3D extracellular matrix (ECM) differentiates mesenchymal stem cells to diverse lineages of osteogenic and adipogenic cells by providing primary heterogeneity. Molecular analysis shows that left-handed chirality of the ECM enhances the clustering of the mechanosensor Itgα5, while right-handed chirality decreases this effect. These differential adhesion patterns further activate distinct mechanotransduction events involving the contractile state, focal adhesion kinase/extracellular signal-regulated kinase 1/2 cascades, and yes-associated protein/runt-related transcription factor 2 nuclear translocation, which direct heterogeneous differentiation. Moreover, theoretical modeling demonstrates that diverse chirality mechanosensing is initiated by biphasic modes of fibronectin tethering. The findings of chirality-dependent lineage specification of stem cells provide potential strategies for the biogenesis of organisms and regenerative therapies.

Morphology of Embryonic Epidermis: An Empirical Multiscale Biophysics Approach

Between nanoscale genotype and mesoscale phenotype, a cascade of physical and biological events take place that translate coded DNA into biological shape, function, and form. But how does this happen? How do we coarse grain from -omics regulation to quasi-pre-programmed anatomical patterns? Here, we use embryonic mouse epidermis as a specific test case to address these questions. Empirical measurements are acquired using the Molecular Atlas Platform - a multiscale imaging technology for visualizing proteomic data across many orders of magnitude in scale - and DNA-PAINT for highly multiplexed super-resolution imaging of immunofluorescent stained molecular targets. We focus on studying the biomechanics of pattern formation, and the sequence of events leading to stem cell fate specification. Our results lead us to a head-on collision with a fundamental chick-and-egg question: Does genetic programming pattern morphology, or do the material effects of morphology pattern gene expression?

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells.