Stem Cells Translational Medicine最新文献

Hematopoietic stem and progenitor cells are responsible for maintenance of the immune system and can be a source of cells for therapies. A critical step in studying or utilizing hematopoietic cells is subpopulation isolation. FerroBio is an emerging technology that uses a streamlined, semi-automated approach to isolate CD34+ cells, which are highly enriched for hematopoietic stem and progenitors. This technology also results in isolation of bead-free CD34+ cell samples, in contrast to traditional kits where beads persist following isolation. Here, we showed a side-by-side comparison of FerroBio isolated cells with CD34+ cells isolated by traditional column-based kits. We showed that FerroBio yields similar numbers of CD34+ cells with similar viability, yield, and gated purity and higher overall purity compared to control kits. FerroBio isolated similar numbers of progenitor cells but significantly higher stem cells. Ex vivo, cells isolated by FerroBio showed the same ability to form colonies in culture, but FerroBio colony-forming units expanded to a greater extent in liquid culture compared to control. Critically, FerroBio isolated cells had equivalent long-term engraftment capacity with significantly better intermediate-term engraftment compared to control in mouse models of transplantation. Based on microscopy images showing altered morphology co-localized with beads, we inferred that the persistence of magnetic microbeads may be associated with the observed differences. These data demonstrated that specific subpopulations of progenitors from FerroBio isolated CD34+ cells have better potency compared to cells isolated with column-based kits. Thus, FerroBio is a viable strategy for isolating CD34+ cells for research and potentially translational utility.

Objective: The development of cartilage tissue engineering offers a promising strategy for effective cartilage regeneration. This study aims to synthesize scaffold-free tissue-engineered cartilage (TEC) using articular chondrocytes (ACs), costal chondrocytes (CCs), bone marrow mesenchymal stem cells (BMSCs), and synovial-derived mesenchymal stromal cells (SDSCs) as seed cells, comparing their cartilage matrix production in vitro and repair capabilities in vivo to guide seed cell selection.

Methods: ACs, CCs, BMSCs, and SDSCs were seeded at high density (4.0 × 105 cells/cm2) in 24-well plates with 0.2 mM Ascorbate 2-phosphate and cultured to form scaffold-free TEC. In vitro, we performed real-time polymerase chain reaction (RT-qPCR), biological analyses, and histochemical staining to assess structural characteristics and cartilage-related gene expression. In vivo, a rat model of knee cartilage defect was used to evaluate tissue repair capabilities, followed by histological assessments and statistical analysis.

Results: In vitro, ACs and CCs showed significantly higher GAG/DNA ratios, with CCs exhibiting the highest chondrogenic gene expression. Histological analysis revealed AC and CC TECs were positive for Safranin-O, Alcian blue, and Col2 staining. In vivo, CC-derived TEC maintained a uniform distribution of Col2, while BMSCs and SDSCs primarily showed Col1 distribution, demonstrating better integration with cartilage and in situ differentiation by 12 weeks.

Conclusion: ACs and CCs excelled in cartilage matrix synthesis; however, AC TEC exhibited phenotypic instability. CC TEC sustained a hyaline chondrocyte phenotype but had later connectivity issues, while BMSCs and SDSCs integrated better but primarily produced fibrous tissue.

Background: Stem cell therapy has emerged as a promising approach for treating critical limb ischemia (CLI), a condition caused by atherosclerosis that results in reduced blood flow and limb necrosis. However, the underlying therapeutic mechanisms involving factors secreted from stem cells are still in the early stages of exploration. This study focuses on investigating the tissue regenerative effects of interleukin-8 (IL8) secreted from cell spheroids.

Methods: Human adipose-derived stem cells (hASCs) were cultured on FGF2-tethered surfaces to form spheroid (FECS-Ad). A murine CLI model was established through femoral artery dissection, followed by the injection of various treatments, including PBS, hASC, FECS-Ad, IL8-silenced FECS-Ad, and recombinant IL8.

Results: Comparative analyses revealed that FECS-Ad injection resulted in a higher percentage of salvaged limbs, but these effects were attenuated when IL8 was silenced in FECS-Ad. Immunofluorescence staining, flow cytometry analysis and RT-qPCR of M1 and M2 macrophage markers demonstrated that IL8 has the ability to polarize macrophages to M2 type. Notably, FECS-Ad injection reduced apoptotic markers (caspase 8 and TUNEL) in ischemic tissues, whereas IL8 knockdown in FECS-Ad increased the proportion of apoptotic cells. FECS-Ad injected tissues showed larger regenerating muscle fibers with centrally located nuclei. Knockdown of IL8 in FECS-Ad decreased the area and size of regenerating muscle fibers.

Conclusions: Our findings underscore the dual role of IL8 in safeguarding muscle tissues from degeneration and orchestrating immunomodulatory effects by finely tuning tissue inflammation and macrophage polarization. This study highlights IL8 as a pivotal paracrine factor contributing to tissue regeneration in the context of stem cell therapy for CLI.

Within the central nervous system (CNS), mitochondria serve as vital energy sources for neurons, glial cells, and vascular functions, maintaining intracellular metabolic balance. Recent studies involving cellular models, rodents, and humans reveal that metabolically active mitochondria can be released into the extracellular space, playing roles in intercellular communication within the CNS. When taken up by neurons, these extracellular mitochondria may provide neuroprotective effects. Conversely, damaged mitochondria and their released components during severe tissue injury or inflammation can contribute to neurodegenerative processes. Thus, mitochondria secreted under pathological conditions in the CNS hold promise as biomarkers indicative of recovery. Additionally, transplantation of external mitochondria shows potential as a therapeutic approach for various CNS disorders. This mini review focuses on recent advances in the transfer of mitochondria between cells, the use of extracellular mitochondria as biomarkers, and the prospects of mitochondria transplantation from experimental research to clinical application, particularly in diseases like stroke.

Tissue dissociation into single-cell suspensions is a critical technique for cell therapy manufacturing, single-cell analysis, and downstream processing. The process is traditionally carried out via enzymatic and mechanical dissociation of the tissue using standard laboratory techniques, but there have also been efforts made to translate these techniques onto microfluidic devices, as well as efforts into performing nonenzymatic digestion. Conventional methods face a number of challenges regarding viability, yield, long processing times, as well as the potential for the processing to create artifacts that can distort downstream analyses. In this review, we discuss the current state-of-the-art technology, go over advancements made in recent years to improve technologies and protocols related to tissue dissociation, and then consider the future of the technique, highlighting ways in which we envision it could be improved.

Autologous fat grafting (AFG), characterized by a broad tissue source and absence of immune rejection, is extensively utilized in plastic surgery. Despite its advantages, AFG is frequently challenged by a high rate of fat resorption and limited volume retention. Recent studies have increasingly focused on integrating platelet-related preparations with adipose tissue to enhance graft survival rates. These investigations have consistently demonstrated the beneficial effects of platelets and their derivatives on adipose-derived stem cells (ADSCs), facilitating improved outcomes in fat transplantation. Nevertheless, the precise mechanisms governing the interaction between platelets and ADSCs remain insufficiently understood. We investigate the potential of platelets to augment the antioxidant stress capacity of ADSCs through mitochondrial transfer, thereby contributing to enhanced fat graft viability. Experimental results revealed that platelets significantly promoted ADSC proliferation, migration, metabolic activity, and mitochondrial function. Co-culture of oxidative stress-induced ADSCs with platelets resulted in improved cell viability and a marked reduction in reactive oxygen species (ROS) levels. The mitochondrial transfer from platelets to ADSCs, confirmed via fluorescent labeling, played a pivotal role in restoring mitochondrial function and decreasing glucose consumption under stress conditions. Furthermore, in a murine subcutaneous fat graft model, platelets exhibited a protective effect during the early oxidative stress phase, as evidenced by reduced ROS and malondialdehyde levels, increased glutathione expression, attenuated fibrosis, enhanced graft vascularization, and improved long-term survival. These findings suggest that platelet-mediated mechanisms, including mitochondrial transfer, may contribute to protecting ADSCs and improving fat graft outcomes.

Tissue engineering for cardiovascular implants has largely utilized primary human cells to generate human tissue-engineered matrices (hTEMs). However, due to donor-to-donor variability and limited passage numbers, a more robust alternative to primary cells would be beneficial. To overcome these limitations, we have defined a new differentiation protocol for human-induced pluripotent stem cells (hiPSCs) into isogeneic cardiac fibroblast-like cells (iCFs) using animal sera-free and chemically defined methods. Morphology, extracellular matrix (ECM) deposition, and global transcriptomics revealed similarity between iCFs and primary human cardiac fibroblasts. Additionally, by overexpressing specific ECM and ECM-related proteins through gene-editing approaches, the ECM composition can be modulated as a building block to create "designer" next-generation hTEMs. Proteomics of gene-edited iCF-derived hTEMs demonstrated an increase in proteins involved in collagen and elastic fiber assembly. Furthermore, analysis of gene-edited iCF-derived hTEM mechanical functionality through biaxial mechanical testing exhibited increased collagen function, attributed to increased crosslinking and maturation. In sum, we have combined hiPSC technology with genome engineering to lay the foundation for next-generation tissue engineering applications by generating a novel cell source, gene-edited iCFs, that are able to modulate the composition as well as the functional mechanics of hTEMs.

Background: Pulmonary endothelial dysfunction with increased capillary permeability is a key aspect in the pathogenesis of acute respiratory distress syndrome (ARDS). It has been demonstrated that mesenchymal stromal cells (MSC) can modulate host cells through mitochondrial transfer. Although mitochondrial transplantation is a promising treatment strategy for conditions underpinned by mitochondrial dysfunction, its therapeutic potential in ARDS has not been sufficiently investigated. Herein, we tested the potential of MSC mitochondrial transplantation to restore functionality of the pulmonary endothelium in pre-clinical models of ARDS.

Methods: Mitochondria (mt) derived from human bone-marrow MSC were isolated and immediately used for transplantation to primary human pulmonary microvascular endothelial cells (HPMEC) in the presence of Escherichia coli lipopolysaccharide (LPS) or plasma samples from ARDS patients classified into hypo- and hyper-inflammatory phenotypes. Mitochondrial function, inflammatory status, and barrier integrity of HPMEC were assessed at 24 h. LPS- challenged mice were treated with MSC-mt intravenously, and the severity of lung injury and inflammatory response were evaluated.

Results: Exposure to LPS or ARDS plasma induced endothelial hyperpermeability associated with mitochondrial dysfunction. MSC-mt were readily internalized by HPMEC without cytotoxicity or inflammatory response, mitigating mitochondrial dysfunction and restoring barrier integrity. In vivo, administration of MSC-mt alleviated lung injury, reduced inflammatory cell infiltration in the alveoli and increased VE-cadherin mRNA levels in the lung tissue, indicating restoration of the alveolar-capillary barrier integrity.

Conclusion: This study demonstrated MSC mitochondrial transplantation as a promising therapeutic approach for treatment of endothelial dysfunction in the context of acute inflammation. Further exploration of mitochondrial transplantation in ARDS is warranted.

Objective: Articular cartilage has limited regenerative capacity due to its lack of innervation, vascularization, and lymphatic vessels. As cartilage is devoid of nerves, injuries often go unnoticed until degeneration leads to pain, reduced function, and ultimately osteoarthritis (OA). Treatment options for cartilage injury, both surgical and nonsurgical, depend on factors like defect size, shape, depth, location, and patient age. Stem cells, particularly their ability to differentiate into chondrocytes, hold promise for cartilage repair, but no therapies have yet gained clinical approval. Recently, induced pluripotent stem cells (iPSCs) have emerged as a potential solution for cartilage regeneration. However, post-transplantation tumorigenesis remains a significant concern. To mitigate this risk, robust quality and safety protocols are needed, alongside safety mechanisms to control iPSC behavior after transplantation.

Design: The iCaspase9 (iCasp9) cell suicide system offers a promising solution, enabling selective elimination of genetically modified cells via apoptosis. We previously demonstrated that the efficiency of iCasp9-mediated killing increases in a p21 mutant background. Since p21 mutations also enhance cartilage repair, we investigated iCasp9-engineered p21-/- and wildtype (p21+/+) iPSCs in a mouse cartilage injury model.

Results: Without iCasp9 activation, both p21-/- and p21+/+ iPSCs formed tumors post-transplantation. In contrast, mice treated with the iCasp9 activator AP20187 showed no tumors. Both p21-/- and p21+/+ iPSCs demonstrated similar cartilage regeneration.

Conclusions: These findings suggest that iCasp9-mediated elimination of iPSCs can effectively mitigate tumor risks while preserving their therapeutic potential for cartilage repair.

High-dose chemotherapy and consecutive autologous stem cell transplantation (ASCT) remain the backbone of treatment for transplant-eligible patients of Multiple Myeloma (MM). However, patients are still at high risk of relapse or treatment-related complications. Hence, by understanding the function of hematopoietic stem and progenitor cells (HSPCs) from MM patients in more detail, transplant outcomes in MM patients might be further improved. We combine in our study functional analyses of the potential of HSPCs from newly diagnosed (NDMM) and chemotherapy treated MM patients in a xenotransplant model system with in depth single cells sequencing analysis to provide novel data that might inform clinical routine to improve the outcome of ASCT in MM. Our data demonstrate that (i) HSPCs from treated MM patients are indeed significantly impaired in their overall reconstitution potential and provide a reduced level of B-cells in comparison to HSPCs from age-matched healthy donors and NDMM patients. (ii) We further demonstrate that CD34+ HSPCs acquire a high-risk MM expression profile signature upon induction treatment, which likely adds to the risk of relapse. This high-risk MM expression profile signature relies within CD34+ HSPCs primarily in granulocyte/macrophage progenitors (GMPs), megakaryocyte Erythroid Progenitors (MEPs) and monocytes, while hematopoietic stem cells (HSCs) stay unaffected by transcriptional changes. These data suggest that the elimination of myeloid progenitors and more mature monocytes (likely by purification for HSCs) in HSPCs harvests from treated MM patients for subsequent ASCT might improve transplant outcomes by avoiding re-infusion of cells with a dysregulated and disease-linked transcriptional program.