Endogenous Cells Research Articles

Structural reconstruction of mouse acute aortic dissection by intravenously administered human Muse cells without immunosuppression

BackgroundStanford type B-acute aortic dissection (type B-AAD) is often life-threatening without invasive surgery. Multilineage-differentiating stress enduring cell (Muse cells), which comprise several percent of mesenchymal stem cells (MSCs), are endogenous pluripotent-like stem cells that selectively home to damaged tissue and replace damaged/apoptotic cells by in-vivo differentiation.MethodsMortality, aortic diameter expansion, cell localization, cell differentiation, and inflammation of the dissected aorta were evaluated in type B-AAD model mice intravenously injected with human-Muse cells, -elastin-knockdown (KD)-Muse cells, -human leukocyte antigen-G (HLA-G)-KD-Muse cells, or MSCs, all without immunosuppressant.ResultsHere, we show the Muse (50,000 cells) group has a lower incidence of aortic rupture and mortality of AAD compared with the MSC-50K (50,000 human-MSCs) and vehicle groups. Spectrum computed tomography in-vivo dynamics and 3-dimensional histologic analyses demonstrate that Muse cells more effectively home to the AAD tissue and survive for 8 weeks in the Muse group than in the MSC-750K (750,000 human-MSCs containing 50,000 Muse cells) group. Homing of Muse cells is impeded in the HLA-G-KD-Muse (50,000 cells) group. Differentiation of homed Muse cells into CD31(+) and alpha-smooth muscle actin (+) cells, production and reorganization of elastic fibers in the AAD tissue, and suppression of diameter expansion are greater in the Muse group than in the MSC-750K and elastin-KD-Muse (50,000 cells) groups.ConclusionsIntravenously administered Muse cells reconstruct the dissected aorta and improve mortality and diameter enlargement rates. Moreover, small doses of purified Muse cells are more effective than large doses of MSCs. HLA-G is suggested to contribute to the successful survival and homing of Muse cells.

Endogenous cell membrane interactome mapping for the GLP-1 receptor in different cell types.

The GLP-1 receptor, one of the most successful drug targets for the treatment of type 2 diabetes and obesity, is known to engage multiple intracellular signaling proteins. However, it remains less explored how the receptor interacts with proteins on the cell membrane. Here, we present a ligand-based proximity labeling approach to interrogate the native cell membrane interactome for the GLP-1 receptor after agonist simulation. Our study identified several unreported putative cell membrane interactors for the endogenous receptor in either a pancreatic β cell line or a neuronal cell line. We further uncovered new regulators of GLP-1 receptor-mediated signaling and insulinotropic responses in β cells. Additionally, we obtained a time-resolved cell membrane interactome map for the receptor in β cells. Therefore, our study provides a new approach that is generalizable to map endogenous cell membrane interactomes for G-protein-coupled receptors to decipher the molecular basis of their cell-type-specific functional regulation.

Sphingosine Kinase 1 Aggravates Liver Fibrosis by Mediating Macrophage Recruitment and Polarization

Sphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells and hepatic stellate cells in liver fibrosis. Here, we aim to investigate the roles of SphK1 on hepatic macrophage recruitment and polarization in liver fibrosis. Liver fibrosis was induced by carbon tetrachloride in wild-type and SphK1-/- mice to study the recruitment and polarization of macrophages. The effects of SphK1 originated from macrophages or other liver cell types on liver fibrosis were further strengthened by bone marrow transplantation. The direct effects of SphK1 on macrophage polarization were also investigated invitro. Expression analysis of SphK1 and macrophage polarization index was conducted with human liver samples. SphK1 deletion attenuated the recruitment of hepatic macrophages along with reduced M1 and M2 polarization in mice induced by carbon tetrachloride. SphK1 deficiency in endogenous liver cells attenuated macrophage recruitment via C-C motif chemokine ligand 2. Macrophage SphK1 activated the ASK1-JNK1/2-p38 signaling pathway to promote M1 polarization. Furthermore, macrophage SphK1 downregulated small ubiquitin-like modifier-specific peptidase1 to decrease de-SUMOylation of Kruppel-like factor 4 to promote M2 polarization. Finally, we confirmed that SphK1 expression was elevated and positively correlated with macrophage M1 and M2 polarization in human fibrosis livers. Our findings demonstrated that SphK1 aggravated liver fibrosis by promoting macrophage recruitment and M1/M2 polarization. SphK1 in macrophages is a potential therapeutic target for the treatment of liver fibrosis.

Engineered Small Extra-Cellular Vesicles for Endogenous Mesenchymal Stem Cells Recruitment and in situ Periodontal Tissue Regeneration

Engineered Small Extra-Cellular Vesicles for Endogenous Mesenchymal Stem Cells Recruitment and in situ Periodontal Tissue Regeneration

NY-ESO-1 antigen: A promising frontier in cancer immunotherapy.

Significant strides have been made in identifying tumour-associated antigens over the past decade, revealing unique epitopes crucial for targeted cancer therapy. Among these, the New York esophageal squamous cell carcinoma (NY-ESO-1) protein, a cancer/testis antigen, stands out. This protein is presented on the cell surface by major histocompatibility complex class I molecules and exhibits restricted expression in germline cells and various cancers, marking it as an immune-privileged site. Remarkably, NY-ESO-1 serves a dual role as both a tumour-associated antigen and its own adjuvant, implying a potential function as a damage-associated molecular pattern. It elicits strong humoural immune responses, with specific antibody frequencies significantly correlating with disease progression. These characteristics make NY-ESO-1 an appealing candidate for developing effective and specific immunotherapy, particularly for advanced stages of disease. In this review, we provide a comprehensive overview of NY-ESO-1 as an immunogenic tumour antigen. We then explore the diverse strategies for targeting NY-ESO-1, including cancer vaccination with peptides, proteins, DNA, mRNA, bacterial vectors, viral vectors, dendritic cells and artificial adjuvant vector cells, while considering the benefits and drawbacks of each strategy. Additionally, we offer an in-depth analysis of adoptive T-cell therapies, highlighting innovative techniques such as next-generation NY-ESO-1 T-cell products and the integration with lymph node-targeted vaccines to address challenges and enhance therapeutic efficacy. Overall, this comprehensive review sheds light on the evolving landscape of NY-ESO-1 targeting and its potential implications for cancer treatment, opening avenues for future tailored directions in NY-ESO-1-specific immunotherapy. HIGHLIGHTS: Endogenous immune response: NY-ESO-1 exhibited high immunogenicity, activating endogenous dendritic cells, T cells and B cells. NY-ESO-1-based cancer vaccines: NY-ESO-1 vaccines using protein/peptide, RNA/DNA, microbial vectors and artificial adjuvant vector cells have shown promise in enhancing immune responses against tumours. NY-ESO-1-specific T-cell receptor-engineered cells: NY-ESO-1-targeted T cells, along with ongoing innovations in engineered natural killer cells and other cell therapies, have improved the efficacy of immunotherapy.

Stem cells, cell therapies, and bioengineering in lung biology and diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Evaluation of the Composite Skin Patch Loaded with Bioactive Functional Factors Derived from Multicellular Spheres of EMSCs for Regeneration of Full-thickness Skin Defects in Rats.

Transplantation of stem cells/scaffold is an efficient approach for treating tissue injury including full-thickness skin defects. However, the application of stem cells is limited by preservation issues, ethical restriction, low viability, and immune rejection in vivo. The mesenchymal stem cell conditioned medium is abundant in bioactive functional factors, making it a viable alternative to living cells in regeneration medicine. Nasal mucosa-derived ecto-mesenchymal stem cells (EMSCs) of rats were identified and grown in suspension sphere-forming 3D culture. The EMSCs-conditioned medium (EMSCs-CM) was collected, lyophilized, and analyzed for its bioactive components. Next, fibrinogen and chitosan were further mixed and cross-linked with the lyophilized powder to obtain functional skin patches. Their capacity to gradually release bioactive substances and biocompatibility with epidermal cells were assessed in vitro. Finally, a full-thickness skin defect model was established to evaluate the therapeutic efficacy of the skin patch. The EMSCs-CM contains abundant bioactive proteins including VEGF, KGF, EGF, bFGF, SHH, IL-10, and fibronectin. The bioactive functional composite skin patch containing EMSCs-CM lyophilized powder showed the network-like microstructure could continuously release the bioactive proteins, and possessed ideal biocompatibility with rat epidermal cells in vitro. Transplantation of the composite skin patch could expedite the healing of the full-thickness skin defect by promoting endogenous epidermal stem cell proliferation and skin appendage regeneration in rats. In summary, the bioactive functional composite skin patch containing EMSCs-CM lyophilized powder can effectively accelerate skin repair, which has promising application prospects in the treatment of skin defects.

A human organoid drug screen identifies α2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration.

Directed differentiation of stem cells toward chondrogenesis invitro and in situ to regenerate cartilage suffers from off-target differentiation and hypertrophic tendency. Here, we generated a cartilaginous organoid system from human expanded pluripotent stem cells (hEPSCs) carrying a COL2A1mCherry and COL10A1eGFP double reporter, enabling real-time monitoring of chondrogenesis and hypertrophy. After screening 2,040 FDA-approved drugs, we found that α-adrenergic receptor (α-AR) antagonists, especially phentolamine, stimulated chondrogenesis but repressed hypertrophy, while α2-AR agonists reduced chondrogenesis and induced hypertrophy. Phentolamine prevented cartilage degeneration in hEPSC cartilaginous organoid and human cartilage explant models and stimulated microfracture-activated endogenous skeletal stem cells toward hyaline-like cartilage regeneration without fibrotic degeneration in situ. Mechanistically, α2-AR signaling induced hypertrophic degeneration via cyclic guanosine monophosphate (cGMP)-dependent secretory leukocyte protease inhibitor (SLPI) production. SLPI-deleted cartilaginous organoid was degeneration resistant, facilitating large cartilage defect healing. Ultimately, targeting α2-AR/SLPI was a promising and clinically feasible strategy to regenerate cartilage via promoting chondrogenesis and repressing hypertrophy.

Endogenous Adult Telomerase Positive Stem Cells Increase in Equine Peripheral Blood Following Exercise

Stout, et al., 2007 reported a base-line level of primitive endogenous adult Telomerase Positive Stem Cells (aTPSCs) circulating within adult porcine peripheral blood which increased dramatically, i.e., 23.5-fold, after just 90 minutes of trauma. Ongoing studies from our laboratory have shown the presence of similar primitive stem cells in adult equine blood and that the base-line levels of stem cells varied according to the particular breed of horse. The current study was undertaken to test the hypothesis that aTPSCs would increase in number in the peripheral blood following stress in the same horse. Stress in this study was defined as moderate exercise, i.e., 10 minutes of cantering. Blood withdrawal followed the guidelines of Fort Valley State University’s IACUC. Adult horses had their blood withdrawn immediately prior to and immediately after exercising. The blood was obtained by venipuncture. Isolated aTPSCs were assayed by trypan blue staining, CEA-CAM-1 for totipotent stem cells and SSEA-4 for pluripotent stem cells. This study demonstrates that moderate exercise will increase the level of these primitive pluripotent stem cells in the blood of adult equines. Studies are ongoing to address their functional significance during injury and repair.

Combinatorial Nutraceutical Supplement Pill (CNSP) stimulates naïve adult telomerase positive stem cells In-Situ to reverse signs and symptoms in multiple human health concerns

Previous IRB-approved studies demonstrated that a rare population of endogenous adult telomerase positive stem cells (aTPSCs), isolated and activated ex vivo, displayed a 100% safety record for reinfusion and an 86% efficacy at reversing signs and symptoms in 97 individuals in 18 disease states. Recent studies demonstrated that these rare aTPSCs could also be activated in vivo by ingestion of a combinatorial nutraceutical supplement pill (CNSP). With IRB permission, CNSP was originally developed for an individual too fragile to undergo the fresh isolate aTPSC technologies. Without any other medical intervention, ingestion of CNSP for 12 months raised his ejection fraction from <10% to about 50%. Based on the results from his study (n=1) [100% safe and 100% efficacious], we received IRB permission to expand the number of clinical trials to determine the effectiveness of using CNSP for other health concerns. Herein, are the preliminary results from 40 people in 28 health concerns utilizing CNSP only and CNSP with other treatment modalities, to affect a better quality of life. Participants were assessed in phase-0, open access, open ended clinical trials, for 1-4+ years. Participants in all 28 clinical trials demonstrated 100% safety and 100% efficacy at reversing signs and symptoms in their respective health concerns, giving them an apparent better quality of life.

Secreted GDF15 maintains transcriptional responses during DNA damage-mediated senescence in human beta cells.

Type 1 diabetes (T1D) is a chronic metabolic disease resulting from an autoimmune destruction of pancreatic beta cells. Beta cells activate various stress responses during the development of T1D, including senescence, which involves cell cycle arrest, prosurvival signaling, and a proinflammatory secretome termed the senescence-associated secretory phenotype (SASP). We previously identified growth and differentiation factor 15 (GDF15) as a major SASP factor in human islets and human EndoC-βH5 beta cells in a model of DNA damage-mediated senescence that recapitulates features of senescent beta cells in T1D. Soluble GDF15 has been shown to exert protective effects on human and mouse beta cells during various forms of stress relevant to T1D; therefore, we hypothesized that secreted GDF15 may play a prosurvival role during DNA damage-mediated senescence in human beta cells. We found that elevated GDF15 secretion was associated with endogenous senescent beta cells in an islet preparation from a T1D donor, supporting the validity of our DNA damage model. Using antibody-based neutralization, we found that secreted endogenous GDF15 was not required for senescent human islet or EndoC cell viability. Rather, neutralization of GDF15 led to reduced expression of specific senescence-associated genes, including GDF15 itself and the prosurvival gene BCL2-like protein 1 (BCL2L1). Taken together, these data suggest that SASP factor GDF15 is not required to sustain senescent human islet viability, but it is required to maintain senescence-associated transcriptional responses.NEW & NOTEWORTHY Beta cell senescence is an emerging contributor to the pathogenesis of type 1 diabetes, but candidate therapeutic targets have not been identified in human beta cells. In this study, we examined the role of a secreted factor, GDF15, and found that although it is not required to maintain viability during senescence, it is required to fine-tune gene expression programs involved in the senescence response during DNA damage in human beta cells.

Stem cell recruitment polypeptide hydrogel microcarriers with exosome delivery for osteoarthritis treatment

With the accelerated aging tendency, osteoarthritis (OA) has become an intractable global public health challenge. Stem cells and their derivative exosome (Exo) have shown great potential in OA treatment. Research in this area tends to develop functional microcarriers for stem cell and Exo delivery to improve the therapeutic effect. Herein, we develop a novel system of Exo-encapsulated stem cell-recruitment hydrogel microcarriers from liquid nitrogen-assisted microfluidic electrospray for OA treatment. Benefited from the advanced droplet generation capability of microfluidics and mild cryogelation procedure, the resultant particles show uniform size dispersion and excellent biocompatibility. Moreover, acryloylated stem cell recruitment peptides SKPPGTSS are directly crosslinked within the particles by ultraviolet irradiation, thus simplifying the peptide coupling process and preventing its premature release. The SKPPGTSS-modified particles can recruit endogenous stem cells to promote cartilage repair and the released Exo from the particles further enhances the cartilage repair performance through synergistic effects. These features suggest that the proposed hydrogel microcarrier delivery system is a promising candidate for OA treatment.

Xenogenous implanted dental follicle stem cells promote periodontal regeneration through inducing the N2 phenotype of neutrophils

BackgroundPeriodontal tissue loss is the main reason for tooth mobility and loss caused by periodontal disease. Dental follicle stem cells (DFSCs) have significant therapeutic potential in periodontal regeneration, which maybe mainly depends on their potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the periodontal defect repair period.MethodsTo trace and investigate the immunomodulation mechanisms of DFSCs in vivo, DFSCs were engineered (E-DFSCs) using lentiviral vectors expressing CD63-enhanced green fluorescent protein (CD63-EGFP) and β-Actin-mCherry protein (ACTB-mCherry) to exhibit green and red fluorescence. The biological characteristics and functions of E-DFSCs were verified by proliferation, differentiation, and co-culture experiments in vitro. In vivo, the periodontal regeneration capacity of E-DFSCs was detected by implantation of murine periodontal defect model, and the response of innate immune cells was detected at the 1st, 3rd, and 5th days (early stage) and 4th week (late stage) after implantation.ResultsIn vitro assessments showed that E-DFSCs retain similar properties to their non-engineered counterparts but exhibit enhanced macrophage immunomodulation capability. In mice models, four-week micro-CT and histological evaluations indicated that E-DFSCs have equivalent efficiency to DFSCs in periodontal defect regeneration. At the early stage of repair in mice periodontal defect, fluorescence tracking showed that implanted E-DFSCs might primarily activate endogenous cells through direct contact and indirect actions, and most of these cells are myeloperoxidase-positive neutrophils. Additionally, compared with the control group, the neutrophilic infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type macrophages, fewer TRAP + osteoclasts, and an upregulated OPG/RANKL ratio were detected in the E-DFSC group compared to the control group, which indicated that immune balance tilts towards healing and bone formation.ConclusionThe xenogenous implanted DFSCs can induce the N2 phenotype of neutrophils in the early stage, which can activate the innate immune mechanism of the host to promote periodontal tissue regeneration.

Accelerated cartilage regeneration through immunomodulation and enhanced chondrogenesis by an extracellular matrix hydrogel encapsulating Kartogenin

Articular cartilage is crucial for the functional integrity of joints yet vulnerable to a spectrum of injuries. The adverse immune microenvironment ensuing from injury, coupled with the intrinsic property of the tissue characterized by limited cellularity, poses a significant challenge to the reconstruction of cartilage defect. In this study, an extracellular matrix hydrogel derived from porcine small intestinal submucosa (SIS) with encapsulation of Kartogenin (KGN) has been designed for the cartilage repair and regeneration. The SIS hydrogel, which possessed a three-dimensional porous structure akin to the natural cartilage, has provided a scaffold essential for the progenitor cells engaged in the repair process and sustained release of KGN. As shown by in vitro experiments, the KGN could recruit host endogenous bone marrow-derived mesenchymal stem cells (BMSCs) and induce them to differentiate into chondrocytes, thus enabling in situ cartilage regeneration without cell transplantation. Notably, the bioactive component of SIS was further revealed to possess excellent immunomodulatory capacity to induce the phenotypic shift from M1 to M2 macrophages, which could enhance the chondrogenic differentiation of BMSCs indirectly. Additionally, in vivo experiments showed that the regenerated tissues of the composite SIS hydrogel group were close to the natural hyaline cartilage. Leveraging the combined effects of KGN and SIS hydrogel, this innovative composite material shows great potential as a biomaterial for effective cartilage repair and regeneration.

Treg-enhancing and immunomodulating microgel scaffold promotes cell ingrowth and heart function recovery post-acute myocardial infarction in vivo

The ingrowth of endogenous cells into tissue scaffolds and modulation of Treg population take a pivotal function in restoring the structure and function of heart post-acute myocardial infarction (MI). However, the traditional hydrogels are failed to achieve such functions due to their critically small mesh size and inherent bioinertness. In this study, a Treg-regulated microgel scaffold (GTK-TK-drug) was prepared by inter-microgels assembly of microgels having cyclodextrin (CD) or adamantane (Ad) with a particle size of about 220 μm. The raw materials to obtain the microgels included gelatin methacryloyl (GelMA), reactive oxygen species (ROS)-responsive crosslinking agent poly(ethylene glycol)-thioketal-poly(ethylene glycol) (PEG-TK-PEG), ROS-responsive release prodrug aminooxyacetic acid (PEG-TK-AOA), PEG-CD or PEG-Ad, and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Quantitative real-time PCR (qPCR), cell counting kit-8, and immunofluorescence staining revealed the effectiveness of AOA for the differentiation of T cells into Treg and the ingrowth of cells in vitro and in vivo. During the short treatment of MI in vivo, the levels of plasma inflammatory factors, cardiomyocyte apoptosis and inflammatory cells were reduced significantly. In the long-term animal experiments, the recovery of cardiac function was significantly enhanced as characterized by echocardiography and the degree of myocardial fibrosis compared to the unassembled microgels control.

Paclobutrazol Enhanced Stem Lodging Resistance of Direct-Seeded Rice by Affecting Basal Internode Development.

The objectives of this study were to explore the mechanism of stem mechanical strength in direct-seeded rice (DSR) as affected by paclobutrazol, especially its related endogenous hormone and cell wall component changes in culm tissue and response to the application of paclobutrazol. Field experiments were conducted in Changchun County, Jilin Province, China, by using two japonica rice varieties, Jiyujing and Jijing305, with soaking seeds in paclobutrazol at concentrations of (0 mg L-1, S0; 50 mg L-1; S1; 100 mg L-1; S2; 150 mg L-1, S3; 200 mg L-1, S4) in 2021 and 2022. The results suggest that the application of paclobutrazol increased the grain yield and reduced the lodging rate of DSR. Compared with the S0 treatments, soaking the seeds in paclobutrazol treatments rapidly shortened the length of the basal internode by decreasing the endogenous indole acetic acid (IAA) and gibberellin A3 (GA3) contents in culm tissue. The larger breaking strength (M) was attributed to a higher section modulus (SM) and bending stress (BS). The higher mechanical tissue thickness in culm tissue under paclobutrazol treatments, which was raised by higher endogenous zeatin and zeatin riboside (Z+ZR) content in culm tissue, increased the culm diameter, culm wall thickness, and section modulus (SM) of the internode. Compared with the S0 treatments, soaking the seeds in paclobutrazol treatments increased the cellulose content, lignin content, activities of lignin-related enzymes, and expression of key genes in lignin biosynthesis, as well as resulted in a higher bending stress (BS) to enhance the culm breaking strength (M).

Safety and feasibility of intravenous administration of a single dose of allogenic-Muse cells to treat human cervical traumatic spinal cord injury: a clinical trial

IntroductionSpinal cord injury (SCI) is a devastating injury and remains one of the largest medical and social burdens because of its intractable nature. According to the recent advances in stem cell biology, the possibility of spinal cord regeneration and functional restoration has been suggested by introducing appropriate stem cells. Multilineage-differentiating stress enduring (Muse) cells are a type of nontumorigenic endogenous reparative stem cell. The positive results of Muse cell transplantation for SCI was shown previously. As a first step for clinical application in human SCI, we conducted a clinical trial aiming to confirm the safety and feasibility of intravenously injected donor-Muse cells.MethodsThe study design of the current trial was a prospective, multicenter, nonrandomized, nonblinded, single-arm study. The clinical trial registration number was JRCT1080224764. Patients with a cervical SCI with a neurological level of injury C4 to C7 with the severity of modified Frankel classification B1 and B2 were included. A primary endpoint was set for safety and feasibility. Our protocol was approved by the PMDA, and the trial was funded by the Life Science Institute, Tokyo, Japan. The present clinical trial recruited 10 participants (8 males and 2 females) with an average age of 49.3 ± 21.2 years old. All 10 participants received a single dose of allogenic CL2020 (a total of 15 × 106 cells, 2.1–2.7 × 105 cells/kg of body weight), which is a Muse cell-based product produced from human mesenchymal stem cells, by an intravenous drip.ResultsThere were two reported severe adverse events, both of which were determined to have no causal relationship with Muse cell treatment. The change in the ISNCSCI motor score, the activity of daily living and quality of life scores showed statistically significant improvements compared to those data at the time of CL2020 administration.ConclusionIn the present trial, no safety concerns were identified, and Muse cell product transplantation demonstrated good tolerability. Future clinical trials with appropriate study designs incorporating a control arm will clarify the definitive efficacy of single-dose allogenic Muse cell treatment with intravenous administration to treat SCI.Trial registration: jRCT, JRCT1080224764. Registered 03 July 2019, https://jrct.niph.go.jp/latest-detail/jRCT1080224764.

Single cell RNA sequencing reveals a shift in cell function and maturation of endogenous and infiltrating cell types in response to acute intervertebral disc injury.

Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous and infiltrating IVD cell populations, as well as the potential of resident mesenchymal stem cells (MSCs) for tissue repair. Control and Injured (needle puncture) tail IVDs were extracted from 12 week old female C57BL/6 mice 7 days post injury and clustering analyses, gene ontology, and pseudotime trajectory analyses were used to determine transcriptomic divergences in the cells of the injured IVD, while immunofluorescence was utilized to determine mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that were IVD tissue specific, immune, or vascular cells. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18+ Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology of DEGs suggested that the most upregulated biological pathways were angiogenesis and T cell related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that cells were driven towards increased cell differentiation due to IVD injury in all IVD tissue clusters except for Krt18+ NP which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more IVD differentiated cells profiles with injury and localized distinctly within the IVD. This study strengthens the understanding of heterogeneous IVD cell populations response to injury and identifies targetable MSC populations for future IVD repair studies.

Optics-free reconstruction of 2D images via DNA barcode proximity graphs.

Spatial genomic technologies include imaging- and sequencing-based methods ( 1-3 ). An emerging subcategory of sequencing-based methods relies on a surface coated with coordinate-associated DNA barcodes, which are leveraged to tag endogenous nucleic acids or cells in an overlaid tissue section ( 4-7 ). However, the physical registration of DNA barcodes to spatial coordinates is challenging, necessitating either high density printing of coordinate-specific oligonucleotides or in situ sequencing/probing of randomly deposited, oligonucleotide-bearing beads. As a consequence, the surface areas available to sequencing-based spatial genomic methods are constrained by the time, labor, cost, and instrumentation required to either print, synthesize or decode a coordinate-tagged surface. To address this challenge, we developed SCOPE (Spatial reConstruction via Oligonucleotide Proximity Encoding), an optics-free, DNA microscopy ( 8 ) inspired method. With SCOPE, the relative positions of randomly deposited beads on a 2D surface are inferred from the ex situ sequencing of chimeric molecules formed from diffusing "sender" and tethered "receiver" oligonucleotides. As a first proof-of-concept, we apply SCOPE to reconstruct an asymmetric "swoosh" shape resembling the Nike logo (16.75 × 9.25 mm). Next, we use a microarray printer to encode a "color" version of the Snellen eye chart for visual acuity (17.18 × 40.97 mm), and apply SCOPE to achieve optics-free reconstruction of individual letters. Although these are early demonstrations of the concept and much work remains to be done, we envision that the optics-free, sequencing-based quantitation of the molecular proximities of DNA barcodes will enable spatial genomics in constant experimental time, across fields of view and at resolutions that are determined by sequencing depth, bead size, and diffusion kinetics, rather than the limitations of optical instruments or microarray printers.

Advancing Scaffold-Assisted Modality for In Situ Osteochondral Regeneration: A Shift From Biodegradable to Bioadaptable.

Over the decades, the management of osteochondral lesions remains a significant yet unmet medical challenge without curative solutions to date. Owing to the complex nature of osteochondral units with multi-tissues and multicellularity, and inherently divergent cellular turnover capacities, current clinical practices often fall short of robust and satisfactory repair efficacy. Alternative strategies, particularly tissue engineering assisted with biomaterial scaffolds, achieve considerable advances, with the emerging pursuit of a more cost-effective approach of in situ osteochondral regeneration, as evolving toward cell-free modalities. By leveraging endogenous cell sources and innate regenerative potential facilitated with instructive scaffolds, promising results are anticipated and being evidenced. Accordingly, a paradigm shift is occurring in scaffold development, from biodegradable and biocompatible to bioadaptable in spatiotemporal control. Hence, this review summarizes the ongoing progress in deploying bioadaptable criteria for scaffold-based engineering in endogenous osteochondral repair, with emphases on precise control over the scaffolding material, degradation, structure and biomechanics, and surface and biointerfacial characteristics, alongside their distinguished impact on the outcomes. Future outlooks of a highlight on advanced, frontier materials, technologies, and tools tailoring precision medicine and smart healthcare are provided, which potentially paves the path toward the ultimate goal of complete osteochondral regeneration with function restoration.