Induction of periodontal ligament-derived mesenchymal stromal cell-like cells from human induced pluripotent stem cells.

Introduction: Periodontal disease is a common oral infection which affects the tooth-supportive tissues directly. Considering the limitation of present regenerative treatments for severe periodontal cases, cytotherapies have been gradually introduced. Human periodontal ligament-derived mesenchymal stromal cells (hPDLMSCs), while identified as one of the promising cell sources for periodontal regenerative therapy, still hold some problems in the clinical application especially their limited life span. To solve the problems, human induced pluripotent stem cells (hiPSCs) are taken into consideration as a robust supply for hPDLMSCs. Methods: The induction of hPDLMSCs was performed based on the generation of neural crest-like cells (NCLCs) from hiPSCs. Fibronectin and laminin were tested as coating materials for NCLCs differentiation when following previous protocol, and the characteristics of induced cells were identified by flow cytometry and RT-qPCR for evaluating the induction efficiency. Subsequently, selected dental ectoderm signaling-related cytokines were applied for hPDLMSCs induction for 14 days, and dental mesenchyme-related genes, dental follicle-related genes and hPDL-related genes were tested by RT-qPCR for the evaluation of differentiation. Results: Compared to the 58% in laminin-coated condition, fibronectin-coated condition had a higher induction efficiency of CD271high cells as 86% after 8-day induction, while the mesenchymal potential of induced NCLCs was similar between two coating materials.It was shown that the gene expressions of dental mesenchyme, dental follicles and hPDL cells were significantly enhanced with the stimulation of the combination with fibroblast growth factor 8b (FGF8b), FGF2, and bone morphogenetic protein 4 (BMP4). Conclusion: FN coating was more effective in NCLCs induction, and the FGF8b+FGF2+BMP4 growth factor cocktail was effective in hPDLMSC-like cell generation. These findings underscored the likely regenerative potential of hiPSCs as an applicable and promising curative strategy for periodontal diseases.

Scale-Up of Coffea canephora Somatic Embryogenesis in Temporary Immersion System.

Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.

Transcriptomic Analysis During the Induction of Somatic Embryogenesis in Coffea canephora.

Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.

Protocol for in vitro co-culture, proliferation, and cell cycle analyses of patient-derived leukemia cells.

Leukemia niche impacts quiescence; however, culturing patient-derived samples ex vivo is technically challenging. Here, we present a protocol for in vitro co-culture of patient-derived xenograft acute lymphoblastic leukemia (PDX-ALL) cells with human mesenchymal stem cells (MSCs). We describe steps for labeling PDX-ALL cells with CellTrace Violet dye to demonstrate MSC-primed PDX-ALL cycling. We then detail procedures to identify MSC-primed G0/quiescent PDX-ALL cells via Hoechst-33342/Pyronin Y live cell cycle analysis. For complete details on the use and execution of this protocol, please refer to Pal et al.1,2.

IKAROS Family Transcription Factors in Lymphocyte Differentiation and Function.

The IKAROS family of transcription factors comprises four zinc-finger proteins (IKAROS, HELIOS, AIOLOS, and EOS), which over the last decades have been established to be critical regulators of the development and function of lymphoid cells. These factors act as homo- or heterodimers and are involved both in gene activation and repression. Their function often involves cross-talk with other regulatory circuits, such as the JAK/STAT, NF-κB, and NOTCH pathways. They control lymphocyte differentiation at multiple stages and are notably critical for lymphoid commitment in multipotent hematopoietic progenitors and for T and B cell differentiation downstream of pre-TCR and pre-BCR signaling. They also control many aspects of effector functions in mature B and T cells. They are dysregulated or mutated in multiple pathologies affecting the lymphoid system, which range from leukemia to immunodeficiencies. In this chapter, we review the molecular and physiological function of these factors in lymphocytes and their implications in human pathologies.

Quantifying Human Naïve B Cell Proliferation Kinetics and Differentiation in Controlled In Vitro Cell Culture.

Division tracking dyes like Cell Trace Violet (CTV) enable the quantification of cell proliferation, division, and survival kinetics of human naïve B cell responses in vitro. Human naïve B cells exhibit distinct responses to different stimuli, with CpG and anti-Ig inducing a T cell-independent (TI) response, while CD40L and IL-21 promote a T cell-dependent (TD) response that induces isotype switching and differentiation into antibody-secreting cells (ASCs). Both stimulation methods yield valuable insights into the intrinsic programming of B cell health within individuals, making them useful for clinical investigations. For instance, quantitative analysis from these B cell populations could reveal biologically meaningful measurements such as the average number of division rounds and the time to cells' fate. Here, we describe a novel in vitro culture setup for CTV-labelled human naïve B cells and a method for obtaining precise time-based data on proliferation, division-linked isotype switching, and differentiation.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Single cell spatial biology over developmental time can decipher pediatric brain pathologies.

Pediatric low grade brain tumors and neurodevelopmental disorders share proteins, signaling pathways, and networks. They also share germline mutations and an impaired prenatal differentiation origin. They may differ in the timing of the events and proliferation. We suggest that their pivotal distinct, albeit partially overlapping, outcomes relate to the cell states, which depend on their spatial location, and timing of gene expression during brain development. These attributes are crucial as the brain develops sequentially, and single-cell spatial organization influences cell state, thus function. Our underlying premise is that the root cause in neurodevelopmental disorders and pediatric tumors is impaired prenatal differentiation. Data related to pediatric brain tumors, neurodevelopmental disorders, brain cell (sub)types, locations, and timing of expression in the developing brain are scant. However, emerging single cell technologies, including transcriptomic, spatial biology, spatial high-resolution imaging performed over the brain developmental time, could be transformational in deciphering brain pathologies thereby pharmacology.

Effects of the MCF-7 Exhausted Medium on hADSC Behaviour.

Stem cells possess the ability to differentiate into different lineages and the ability to self-renew, thus representing an excellent tool for regenerative medicine. They can be isolated from different tissues, including the adipose tissue. Adipose tissue and human adipose-derived stem cells (hADSCs) are privileged candidates for regenerative medicine procedures or other plastic reconstructive surgeries. The cellular environment is able to influence the fate of stem cells residing in the tissue. In a previous study, we exposed hADSCs to an exhausted medium of a breast cancer cell line (MCF-7) recovered at different days (4, 7, and 10 days). In the same paper, we inferred that the medium was able to influence the behaviour of stem cells. Considering these results, in the present study, we evaluated the expression of the major genes related to adipogenic and osteogenic differentiation. To confirm the gene expression data, oil red and alizarin red colorimetric assays were performed. Lastly, we evaluated the expression of miRNAs influencing the differentiation process and the proliferation rate, maintaining a proliferative state. The data obtained confirmed that cells exposed to the medium maintained a stem and proliferative state that could lead to a risky proliferative phenotype.

A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation.

The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

Dentin matrix protein 1 and HUVEC-ECM scaffold promote the differentiation of human dental pulp stem cells into endothelial lineage: implications in regenerative medicine.

Reprograming of the dental pulp somatic cells to endothelial cells is an attractive strategy for generation of new blood vessels. For tissue regeneration, vascularization of engineered constructs is crucial to improve repair mechanisms. In this study, we show that dentin matrix protein 1 (DMP1) and HUVEC-ECM scaffold enhances the differentiation potential of dental pulp stem cells (DPSCs) to an endothelial phenotype. Our results show that the differentiated DPSCs expressed endothelial markers CD31 and VE-Cadherin (CD144) at 7 and 14 days. Expression of CD31 and VE-Cadherin (CD144) were also confirmed by immunofluorescence. Furthermore, flow cytometry analysis revealed a steady increase in CD31 and VE-Cadherin (CD144) positive cells with DMP1 treatment when compared with control. In addition, integrins specific for endothelial cells were highly expressed during the differentiation process. The endothelial cell signature of differentiated DPSCs were additionally characterized for key endothelial cell markers using gene expression by RT-PCR, Western blotting, immunostaining, and RNA-seq analysis. Furthermore, the angiogenic phenotype was confirmed by tubule and capillary sprout formation. Overall, stimulation of DPSCs by DMP1 and use of HUVEC-ECM scaffold promoted their differentiation into phenotypically, transcriptionally, and functionally differentiated bonafide endothelial cells. This study is novel, physiologically relevant and different from conventional strategies.

A New Tissue Engineering Strategy to Promote Tendon-bone Healing: Regulation of Osteogenic and Chondrogenic Differentiation of Tendon-derived Stem Cells.

In the field of sports medicine, repair surgery for anterior cruciate ligament (ACL) and rotator cuff (RC) injuries are remarkably common. Despite the availability of relatively effective treatment modalities, outcomes often fall short of expectations. This comprehensive review aims to thoroughly examine current strategies employed to promote tendon-bone healing and analyze pertinent preclinical and clinical research. Amidst ongoing investigations, tendon-derived stem cells (TDSCs), which have comparatively limited prior exploration, have garnered increasing attention in the context of tendon-bone healing, emerging as a promising cell type for regenerative therapies. This review article delves into the potential of combining TDSCs with tissue engineering methods, with ACL reconstruction as the main focus. It comprehensively reviews relevant research on ACL and RC healing to address the issues of graft healing and bone tunnel integration. To optimize tendon-bone healing outcomes, our emphasis lies in not only reconstructing the original microstructure of the tendon-bone interface but also achieving proper bone tunnel integration, encompassing both cartilage and bone formation. In this endeavor, we thoroughly analyze the transcriptional and molecular regulatory variables governing TDSCs differentiation, incorporating a retrospective analysis utilizing single-cell sequencing, with the aim of unearthing relevant signaling pathways and processes. By presenting a novel strategy rooted in TDSCs-driven osteogenic and chondrogenic differentiation for tendon-bone healing, this study paves the way for potential future research avenues and promising therapeutic applications. It is anticipated that the findings herein will contribute to advancing the field of tendon-bone healing and foster the exploration of TDSCs as a viable option for regenerative therapies in the future.

Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit.

Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

Fatty acid oxidation in immune function.

Cellular metabolism is a crucial determinant of immune cell fate and function. Extensive studies have demonstrated that metabolic decisions influence immune cell activation, differentiation, and cellular capacity, in the process impacting an organism's ability to stave off infection or recover from injury. Conversely, metabolic dysregulation can contribute to the severity of multiple disease conditions including autoimmunity, alloimmunity, and cancer. Emerging data also demonstrate that metabolic cues and profiles can influence the success or failure of adoptive cellular therapies. Importantly, immunometabolism is not one size fits all; and different immune cell types, and even subdivisions within distinct cell populations utilize different metabolic pathways to optimize function. Metabolic preference can also change depending on the microenvironment in which cells are activated. For this reason, understanding the metabolic requirements of different subsets of immune cells is critical to therapeutically modulating different disease states or maximizing cellular function for downstream applications. Fatty acid oxidation (FAO), in particular, plays multiple roles in immune cells, providing both pro- and anti-inflammatory effects. Herein, we review the major metabolic pathways available to immune cells, then focus more closely on the role of FAO in different immune cell subsets. Understanding how and why FAO is utilized by different immune cells will allow for the design of optimal therapeutic interventions targeting this pathway.

Melatonin via MTNR1B regulates METTL3 to protect ileum cell differentiation.

Intestinal stem cells rapidly differentiate into various epithelial cells, playing a crucial role in maintaining intestinal homeostasis. Melatonin, a known endogenous molecule with anti-inflammatory and antioxidant properties, has its potential efficacy in ileum stem cells differentiation not fully understood to date. This study indicates that melatonin suppresses ileum inflammation and maintains normal differentiation of ileum stem cells through MTNR1B. Subsequent outcomes following treatment with MTNR1B inhibitors further substantiate these findings. Additionally, overexpression of METTL3 protein appears to be a potential instigator for promoting ileum inflammation and disruptions in cell differentiation. Treatment with the METTL3 inhibitor SAH significantly inhibits ileum inflammation and Wnt/ß-catenin activity, thereby sustaining normal cellular differentiation functions. In summary, this study showed that melatonin may improve ileum inflammation and maintain cell differentiation functions by inhibiting abnormal METTL3 expression via MTNR1B.

Expression patterns of keratin family members during tooth development and the role of keratin 17 in cytodifferentiation of stratum intermedium and stellate reticulum.

Keratins are typical intermediate filament proteins of the epithelium that exhibit highly specific expression patterns related to the epithelial type and stage of cellular differentiation. They are important for cytoplasmic stability and epithelial integrity and are involved in various intracellular signaling pathways. Several keratins are associated with enamel formation. However, information on their expression patterns during tooth development remains lacking. In this study, we analyzed the spatiotemporal expression of keratin family members during tooth development using single-cell RNA-sequencing (scRNA-seq) and microarray analysis. scRNA-seq datasets from postnatal Day 1 mouse molars revealed that several keratins are highly expressed in the dental epithelium, indicating the involvement of keratin family members in cellular functions. Among various keratins, keratin 5 (Krt5), keratin 14 (Krt14), and keratin 17 (Krt17) are highly expressed in the tooth germ; KRT17 is specifically expressed in the stratum intermedium (SI) and stellate reticulum (SR). Depletion of Krt17 did not affect cell proliferation in the dental epithelial cell line SF2 but suppressed their differentiation ability. These results suggest that Krt17 is essential for SI cell differentiation. Furthermore, scRNA-seq results indicated that Krt5, Krt14, and Krt17 exhibited distinct expression patterns in ameloblast, SI, and SR cells. Our findings contribute to the elucidation of novel mechanisms underlying tooth development.

Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors.

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

The dynamic expression of YAP is essential for the development of male germ cells derived from human embryonic stem cells.

YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.

Protocol for the derivation and alveolar type 2 differentiation of late-stage lung tip progenitors from the developing human lungs.

Molecular and cellular mechanisms of human lung alveolar development are poorly understood due to a lack of in vitro model systems. This protocol details the isolation, derivation, and genetic modification of lung tip epithelial progenitors from human fetal lungs. It includes steps for isolating distal lung epithelial cells, expanding tip progenitor organoids, culturing tip organoids in vitro, and differentiating them into alveolar type 2 cells. This will aid in understanding alveolar differentiation mechanisms and neonatal diseases. For complete details on the use and execution of this protocol, please refer to Lim et al.1.