Cells Tissues Organs最新文献

Introduction: Generating new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are still lacking.

Methods: Transcription factors, ETS2 and ETV2 are key regulators of embryonic vascular development, including lymphatic specification. While ETV2 has been shown to efficiently generate blood endothelial cells, little is known about ETS2 and its role in lymphatic differentiation. Here, we describe a method for rapid and efficient generation of LECs using transcription factors, ETS2 and ETV2.

Results: This approach reproducibly differentiates four diverse hiPSCs into LECs with exceedingly high efficiency. Timely activation of ETS2 was critical, to enable its interaction with Prox1, a master lymphatic regulator. Differentiated LECs express key lymphatic markers, VEGFR3, LYVE-1, and Podoplanin, in comparable levels to mature LECs. The differentiated LECs are able to assemble into stable lymphatic vascular networks in vitro, and secrete key lymphangiocrine, reelin.

Conclusion: Overall, our protocol has broad applications for basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine.

Introduction: Basic helix-loop-helix (bHLH) transcription factors are expressed in various organs and are involved in diverse developmental processes. The mouse atonal homolog 8 (Atoh8), a bHLH transcription factor, plays a crucial role in various developmental processes, especially as a regulator of neurogenesis in the retina. Besides, Atoh8 expression has been observed in the central nervous system. The function of Atoh8 during the postnatal neurogenesis is still unclear.

Methods: This study focuses on elucidating the impact of Atoh8 on postnatal neurogenesis in the brain, particularly in selected regions: the subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB), across different life stages, using male homozygous Atoh8-knockout (M6KO) mice. Our morphometric analysis is based on immunohistochemically labeled markers for neuroblasts (doublecortin) and proliferation (phospho-histone H3, PHH3) as well as pan neuronal markers.

Results: In Atoh8-/- mice, alteration in the postnatal neurogenesis can be observed. Immunohistochemical analysis revealed a significant reduction in doublecortin-positive neuroblasts within the SVZ of neonatal M6KO mice compared to wild-type mice. Interestingly, no differences in cell number and distribution were observed in the subsequent migration of neuroblasts through the RMS to the OB. Proliferating PHH3-positive neuronal progenitor cells were significantly diminished in the proliferation rate in both the SVZ and RMS of neonatal and young M6KO mice. Furthermore, in the glomerular layer of the OB, significantly fewer neurons were detected in the neonatal stage.

Conclusion: In conclusion, Atoh8 emerges as a positive regulator of postnatal neurogenesis in the brain. Its role encompasses the promotion of neuroblast formation, modulation of proliferation rates, differentiation, and maintenance of mature neurons. Understanding the intricacies of Atoh8 function provides valuable insights into the complex regulatory mechanisms governing neurogenesis.

Introduction: To date, there have been no studies conducted on the development of interosseous muscles (IO) in the human hand. This study aimed to investigate the development of these muscles in order to clarify their terminal insertions and their relationship with the metacarpophalangeal joints.

Methods: Serial sections of 25 human specimens (9 embryos and 16 fetuses) between the 7th and 14th weeks of development, sourced from the Collection of the Department of Anatomy and Embryology at UCM Faculty of Medicine, were analyzed bilaterally using a conventional optical microscope.

Results: Our findings revealed that, during the 7th week of development, the metacarpophalangeal interzone mesenchyme extended into the extensor apparatus of the fingers. Furthermore, we observed that the joint capsule and the tendon of the IO derive from the articular interzone mesenchyme. By the end of the 7th week, corresponding to Carnegie stage 21, the myotendinous junction appeared, initiating cavitation of the metacarpophalangeal joint. During the fetal period, the terminal insertions of the IO were identified: both the dorsal interosseous (DI) and palmar interosseous (PI) muscles insert into the metacarpophalangeal joint capsule and establish a connection with the volar plate located at the base of the proximal phalanx and the extensor apparatus. Some muscle fibers also attach to the joint capsule at the level of the proximal synovial cul-de-sac. The functional implications of these findings are discussed within this work.

Conclusion: This study provides the first detailed description of the development of the interosseous muscles in the human hand.

Introduction: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes.

Methods: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription.

Results: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates.

Conclusion: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

Introduction: Endothelial cells (EC) can be generated from porcine-induced pluripotent stem cells (piPSC), but poor efficiency in driving EC differentiation hampers their application and efficacy. Additionally, the culture of piPSC-derived EC (piPSC-EC) on three-dimensional (3D) scaffolds has not been fully reported yet. Here, we report a method to improve the generation of EC differentiation from piPSC and to facilitate their culture on 3D scaffolds, providing a potential resource for in vitro drug testing and the generation of tissue-engineered vascular grafts.

Methods: We initiated the differentiation of piPSC into EC by seeding them on laminin 411 and employing a three-stage protocol, which involved the use of distinct EC differentiation media supplemented with CHIR99021, BMP4, VEGF, and bFGF.

Results: piPSC-EC not only expressed EC markers such as CD31, VE-cadherin, and von Willebrand factor (vWF) but also exhibited an upregulation of EC marker genes, including CD31, CD34, VEGFR2, VE-cadherin, and vWF. They exhibited functional characteristics similar to those of porcine coronary artery endothelial cells (PCAEC), such as tube formation and Dil-Ac-LDL uptake. Furthermore, when cultured on 3D scaffolds, piPSC-EC developed a 3D morphology and were capable of forming an endothelial layer and engineering capillary-like networks, though these lacked lumen structures.

Conclusion: Our study not only advances the generation of EC from piPSC through an inhibitor and growth factor cocktail but also provides a promising approach for constructing vascular network-like structures. Importantly, these findings open new avenues for drug discovery in vitro and tissue engineering in vivo.

Introduction: Localized delivery of angiogenesis-promoting factors such as small molecules, nucleic acids, peptides, and proteins to promote the repair and regeneration of damaged tissues remains a challenge in vascular tissue engineering. Current delivery methods such as direct administration of therapeutics can fail to maintain the necessary sustained release profile and often rely on supraphysiologic doses to achieve the desired therapeutic effect. By implementing a microparticle delivery system, localized delivery can be coupled with sustained and controlled release to mitigate the risks involved with the high dosages currently required from direct therapeutic administration.

Methods: For this purpose, poly(lactic-co-glycolic acid) (PLGA) microparticles were fabricated via anti-solvent microencapsulation and the loading, release, and delivery of model angiogenic molecules, specifically a small molecule, nucleic acid, and protein, were assessed in vitro using microvascular fragments (MVFs).

Results: The microencapsulation approach utilized enabled rapid spherical particle formation and encapsulation of model drugs of different sizes, all in one method. The addition of a fibrin scaffold, required for the culture of the MVFs, reduced the initial burst of model drugs observed in release profiles from PLGA alone. Lastly, in vitro studies using MVFs demonstrated that higher concentrations of microparticles led to greater co-localization of the model therapeutic (miRNA) with MVFs, which is vital for targeted delivery methods. It was also found that the biodistribution of miRNA using the delivered microparticle system was enhanced compared to direct administration.

Conclusion: Overall, PLGA microparticles, formulated and loaded with model therapeutic compounds in one step, resulted in improved biodistribution in a model of the vasculature leading to a future in translational revascularization.

Introduction: Acupuncture has been used for pain management for thousands of years. However, it is largely unclear whether this therapeutic approach can effectively reduce heart failure-associated symptoms, including dyspnea. The hypothesis posited in this study was that acupuncture does indeed aid in the management of such symptoms and was motivated by the following statistics that establish a requisite need for efficient management of dyspnea to improve patient outcomes with heart failure. In 2020, an estimated 6.2 million adults in the USA had a heart failure diagnosis; in 2018, 379,800 death certificates reported heart failure; and the national cost of heart failure in 2012 was approximately USD 30.7 billion.

Methods: The methodology employed to conduct this study involved review of trial data extracted from review of papers pertaining to acupuncture, symptoms of heart failure, and dyspnea, from academic and clinical data repositories subject to various inclusion and exclusion criteria. Of the initial set of 293 studies identified, the resulting inclusion set comprised 30 studies. The analysis conducted revealed that the highest frequency of combined acupuncture points prescribed for the foregoing search criteria were as follows: BL13, BL23, LU9, LU5, Dingchuan, LI4, PC6, and HT7.

Results: A meta-analysis of combined pooled p values for the studies revealed that acupuncture does aid in the management of symptoms of dyspnea and heart failure, subject to various limitations including but not limited to heterogeneity inherent between the studies in the inclusion set that were analyzed. Such limitations underscore the need to restrict generalizations from the conclusions of this study.

Conclusion: The impact and novelty of this research study is its attempt to target the apparent paucity of literature that focuses on the management of dyspnea specifically in the context of heart failure with acupuncture and to bridge the gap of the application of acupuncture research on dyspnea to the cardiovascular context of heart failure. Notwithstanding the meta-analysis undertaken under this review study, further statistical analysis and a pilot study are warranted to consolidate or nullify the results of the research.

Introduction: The influence of mechanical forces generated by stromal cells in the perivascular matrix is thought to be a key regulator in controlling blood vessel growth. Cadherins are mechanosensors that facilitate and maintain cell-cell interactions and blood vessel integrity, but little is known about how stromal cells regulate cadherin signaling in the vasculature. Our objective was to investigate the relationship between mechanical phenotypes of stromal cells with cadherin expression in 3D tissue engineering models of vascular growth.

Methods: Stromal cell lines were subjected to a bead displacement assay to track matrix distortions and characterize mechanical phenotypes in 3D microtissue models. These cells included human ventricular cardiac (NHCF), dermal (NHDF), lung (NHLF), breast cancer-associated (CAF), and normal breast fibroblasts (NBF). Cells were embedded in a fibrin matrix (10 mg/mL) with fluorescent tracker beads; images were collected every 30 min. We also studied endothelial cells (ECs) in co-culture with mechanically active or inactive stromal cells and quantified N-Cad, OB-Cad, and VE-Cad expression using immunofluorescence.

Results: Bead displacement studies identified mechanically active stromal cells (CAFs, NHCFs, NHDFs) that generate matrix distortions and mechanically inactive cells (NHLFs, NBFs). CAFs, NHCFs, and NHDFs displaced the matrix with an average magnitude of 3.17 ± 0.11 μm, 3.13 ± 0.06 μm, and 2.76 ± 0.05 μm, respectively, while NHLFs and NBFs displaced the matrix with an average of 1.82 ± 0.05 μm and 2.66 ± 0.06 μm in fibrin gels. Compared to ECs only, CAFs + ECs as well as NBFs + ECs in 3D co-culture significantly decreased expression of VE-Cad; in addition, Pearson's Correlation Coefficient for N-Cad and VE-Cad showed a strong correlation (>0.7), suggesting cadherin colocalization. Using a microtissue model, we demonstrated that mechanical phenotypes associated with increased matrix deformations correspond to enhanced angiogenic growth. The results could suggest a mechanism to control tight junction regulation in developing vascular beds for tissue engineering scaffolds or understanding vascular growth during developmental processes.

Conclusion: Our studies provide novel data for how mechanical phenotype of stromal cells in combination with secreted factor profiles is related to cadherin regulation, localization, and vascularization potential in 3D microtissue models.

Background: With the elderly population projected to double by 2050, there is an urgent need to address the increasing prevalence of age-related debilitating diseases and ultimately minimize discrepancies between the rising lifespan and stagnant health span. Cellular reprogramming by overexpression of Oct3/4, Klf4, Sox2, and cMyc (OKSM) transcription factors is gaining attention in this context thanks to demonstrated rejuvenating effects in human cell cultures and live mice, many of which can be uncoupled from dedifferentiation and loss of cell identity.

Summary: Here, we review current evidence of the impact of cell reprogramming on established aging hallmarks and the underlying mechanisms that mediate these effects. We also provide a critical assessment of the challenges in translating these findings and, overall, cell reprogramming technologies into clinically translatable antiaging interventions.

Key messages: Cellular reprogramming has the potential to reverse at least partially some key hallmarks of aging. However, further research is necessary to determine the biological significance and duration of such changes and to ensure the safety of cell reprogramming as a rejuvenation approach. With this review, we hope to stimulate new research directions in the quest to extend health span effectively.

Introduction: Sensory nerve endings transmit mechanical stimuli into afferent neural signals and form the basis of proprioception, giving rise to the self-perception of dynamic stability of joints. We aimed to analyze the three-dimensional structure of periarticular corpuscular sensory nerve endings in a carpal ligament to enhance our understanding of their microstructure.

Methods: Two dorsal parts of the scapholunate ligament were excised from two human cadaveric wrist specimens. Consecutive cryosections were stained with immunofluorescence markers protein S100B, neurotrophin receptor p75, protein gene product 9.5 (PGP 9.5), and 4',6-diamidino-2-phenylindole. Three-dimensional images of sensory nerve endings were obtained using confocal laser scanning microscopy, and subsequent analysis was performed using Imaris software.

Results: Ruffini endings were characterized by a PGP 9.5-positive central axon, with a median diameter of 4.63 μm and a median of 25 cells. The p75-positive capsule had a range in thickness of 0.94 μm and 15.5 μm, consisting of single to three layers of lamellar cells. Ruffini endings were significantly smaller in volume than Pacini corpuscles or Golgi-like endings. The latter contained a median of three intracorpuscular structures. Ruffini endings and Golgi-like endings presented a similar structural composition of their capsule and subscapular space. The central axon of Pacini corpuscles was surrounded by S100-positive cells forming the inner core which was significantly smaller than the outer core, which was immunoreactive for p75 and PGP 9.5.

Conclusion: This study reports new data regarding the intricate outer and intracorpuscular three-dimensional morphology of periarticular sensory nerve endings, including the volume, number of cells, and structural composition. These results may form a basis to differ between normal and pathological morphological changes in periarticular sensory nerve endings in future studies.