Number Of Mesenchymal Stem Cells Research Articles

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain

BackgroundHypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain.MethodsHI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay.ResultsIntranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion.ConclusionsThis study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Age-Related Effects on MSC Immunomodulation, Macrophage Polarization, Apoptosis, and Bone Regeneration Correlate with IL-38 Expression.

Mesenchymal stem cells (MSCs) are known to promote tissue regeneration and suppress excessive inflammation caused by infection or trauma. Reported evidence indicates that various factors influence the expression of MSCs' endogenous immunomodulatory properties. However, the detailed interactions of MSCs with macrophages, which are key cells involved in tissue repair, and their regulatory mechanisms are not completely understood. We herein investigated how age-related immunomodulatory impairment of MSCs alters the interaction of MSCs with macrophages during bone healing using young (5-week old) and aged (50-week old) mice. To clarify the relationship between inflammatory macrophages (M1) and MSCs, their spatiotemporal localization at the bone healing site was investigated by immunostaining, and possible regulatory mechanisms were analyzed in vitro co-cultures. Histomorphometric analysis revealed an accumulation of M1 and a decrease in MSC number at the healing site in aged mice, which showed a delayed bone healing. In in vitro co-cultures, MSCs induced M1 apoptosis through cell-to-cell contact but suppressed the gene expression of pro-inflammatory cytokines by soluble factors secreted in the culture supernatant. Interestingly, interleukin 38 (Il-38) expression was up-regulated in M1 after co-culture with MSCs. IL-38 suppressed the gene expression of inflammatory cytokines in M1 and promoted the expression of genes associated with M1 polarization to anti-inflammatory macrophages (M2). IL-38 also had an inhibitory effect on M1 apoptosis. These results suggest that MSCs may induce M1 apoptosis, suppress inflammatory cytokine production by M1, and induce their polarization toward M2. Nevertheless, in aged conditions, the decreased number and immunomodulatory function of MSCs could be associated with a delayed M1 clearance (i.e., apoptosis and/or polarization) and consequent delayed resolution of the inflammatory phase. Furthermore, M1-derived IL-38 may be associated with immunoregulation in the tissue regeneration site.

An Overview of Mesenchymal Stem Cell Heterogeneity and Concentration.

Mesenchymal stem cells (MSCs) are of great interest in cell therapies due to the immunomodulatory and other effects they have after autologous or allogeneic transplantation. In most clinical applications, a high number of MSCs is required; therefore, the isolated MSC population must be expanded in the cell culture until the desired number is reached. Analysing freshly isolated MSCs is challenging due to their rareness and heterogeneity, which is noticeable among donors, tissues, and cell subpopulations. Although the phenotype of MSCs in tissue can differ from those of cultured cells, phenotyping and counting are usually performed only after MSC proliferation. As MSC applicability is a developing and growing field, there is a need to implement phenotyping and counting methods for freshly isolated MSCs, especially in new one-step procedures where isolated cells are implanted immediately without cell culturing. Only by analysing harvested cells can we correctly evaluate such studies. This review describes multilevel heterogeneity and concentrations of MSCs and different strategies for phenotype determination and enumeration of freshly isolated MSCs.

Investigation of the differentiation potential of pericyte cells as an alternative source of mesenchymal stem cells

BackgroundThe mesenchymal stem cells (MSCs) with characterized by their multipotency and capacity to differentiate into various tissue cell types, have led to their incorporation in regenerative medicine research. However, the limited numbers of MSCs in the human body and their diverse differentiation capabilities in tissues highlight the need for exploring alternative regenerative cell sources. In this study, therefore, we conducted molecular level examinations to determine whether pericytes, specialized cell communities situated near blood vessels, could serve as a substitute for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this context, the potential application of pericytes surrounds the vessels when MSCs are insufficient for functional purposes. MethodsThe pericytes utilized in this investigation were derived from the placenta and characterized at the third passage. Similarly, the hBM-MSCs were also characterized at the third passage. The pluripotent properties of the two cell types were assessed at the gene expression level. Thereafter, both pericytes and hBM-MSCs were directed towards adipogenic, osteogenic and chondrogenic differentiation. The cells in both groups were examined on days 7, 14, and, 21 and their differentiation status was compared both immunohistochemically and through gene expression analysis. ResultsUpon comparing the pluripotency characteristics of placental pericytes and hBM-MSCs, it was discovered that there was a substantial upregulation of the pluripotency genes FoxD3, Sox2, ZPF42, UTF1, and, Lin28 in both cell types. However, no significant expression of the genes Msx1, Nr6a1, Pdx1, and, GATA6 was observed in either cell type. It was also noted that pericytes differentiate into adipogenic, osteogenic and, chondrogenic lineages similar to hBM-MSCs. DiscussionAs a result, it has been determined that pericytes exhibit high differentiation and proliferation properties similar to those of MSCs, and therefore can be considered a suitable alternative cell source for regenerative medicine and tissue engineering research, in cases where MSCs are not available or insufficient. It is notable that pericytes have been suggested as a potential substitute in studies where MSCs are lacking.

COMPARISON OF AUTOLOGOUS ADIPOSE-DERIVED TISSUE STROMAL VASCULAR FRACTION (AD-TSVF) BIOMEDICAL INSTRUMENTS FOR THEIR STEM CELL CONTENT

Deriving autologous mesenchymal stem cells (MSCs) from adipose tissues without using enzymes requires sophisticated biomedical instruments. Applied pressure on tissues and cells are adjusted manually although centrifugation and filtration systems are frequently used. The number of derived MSCs therefore could differ between instruments. We compared the number of MSCs obtained from four commercially available devices and our newly designed and produced instrument (A2, B3, L3, M2 and T3). Three-hundred mL of adipose tissue was obtained from a female patient undergoing liposuction using the transillumination solution. Obtained tissue was equally distributed to each device and handled according to the producers' guides. After handling, 3 mL stromal vascular fraction (SVF) was obtained from each device. Freshly isolated SVF was characterized using multi-color flow cytometry (Navios Flow Cytometer, Beckman Coulter, USA). Cell surface antigens were chosen according to IFATS and ISCT. CD31-FITC, CD34-PC5,5, CD73-PE, CD90-PB and CD45-A750 (Backman Coulter, USA) fluorochrome-labeled monoclonal antibodies were assessed. Markers were combined with ViaKrome (Beckman Coulter, USA) to determine cell viability. At least 105 cells were acquired from each sample. A software (Navios EX, Beckman Coulter, USA) was used to create dot plots and to calculate the cell composition percentages. The data was analyzed in the Kaluza 2.1 software package (Beckman Coulter, USA). Graphs were prepared in GraphPad Prism. CD105 PC7/CD31 FITC cell percentages were 23,9%, 13,5%, 24,6%, 11,4% and 28,8% for the A2, B3, L3, M2 and T3 devices, respectively. We conclude that the isolated MSC percentage ranged from 11,4% to 28,8% between devices. The number of MSCs in SVF are key determinants of success in orthobiological treatments. Developing a device should focus on increasing the number of MSCs in the SVF while preserving its metabolic activity.Acknowledgments: Scientific and Technological Research Council of Türkiye (TÜBİTAK)- Technology and Innovation Funding Program Directorate (TEYDEB) funded this project (#321893). Servet Kürümoğlu and Bariscan Önder of Disposet Ltd., Ankara, Türkiye (www.disposet.com) contributed to the industrial design and research studies. Ali Tuncel and Feza Korkusuz are members of the Turkish Academy of Sciences (TÜBA). Nilsu Baysal was funded by the STAR Program of TÜBITAK Grant # 3210893.

Age-Related Alterations in Mesenchymal Stem Cell Function: Understanding Mechanisms and Seeking Opportunities to Bypass the Cellular Aging.

Undoubtedly, mesenchymal stem cells (MSCs) are the most common cell therapy candidates in clinical research and therapy. They not only exert considerable therapeutic effects to alleviate inflammation and promote regeneration, but also show low-immunogenicity properties, which ensure their safety following allogeneic transplantation. Thanks to the necessity of providing a sufficient number of MSCs to achieve clinically efficient outcomes, prolonged in vitro cultivation is indisputable. However, either following long-term in vitro expansion or aging in elderly individuals, MSCs face cellular senescence. Senescent MSCs undergo an impairment in their function and therapeutic capacities and secrete degenerative factors which negatively affect young MSCs. To this end, designing novel investigations to further elucidate cellular senescence and to pave the way toward finding new strategies to reverse senescence is highly demanded. In this review, we will concisely discuss current progress on the detailed mechanisms of MSC senescence and various inflicted changes following aging in MSC. We will also shed light on the examined strategies underlying monitoring and reversing senescence in MSCs to bypass the comprised therapeutic efficacy of the senescent MSCs.

A modified animal-free serum technique for efficient isolation and proliferation of mesenchymal stem cells from Hoffa fat pad.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Vascular Cell Adhesion Molecule-1 Regulates Bone Marrow Mesenchymal Stem Cell Maintenance and Niche Function

Bone marrow (BM) mesenchymal stem cells (MSCs) are rare non-hematopoietic perivascular stromal cells characterized by their unique ability to self-renew and differentiate into bone, cartilage, and fat, ensuring proper skeletal development and maintenance. Different subsets of BM MSCs associate with arteriolar and sinusoidal vessels to form specialized niches that regulate hematopoietic stem cell (HSC) function by secreting high levels of niche factors such as CXC-chemokine ligand 12 (CXCL12), stem cell factor (SCF), and Vascular Cell Adhesion Molecule-1 (VCAM1). VCAM1 is classically expressed on endothelial and stromal cells where it acts as an adhesion molecule that preferentially binds to α4β1 integrin on HSCs and progenitor cells (HSPCs). Deletion of Vcam1 in endothelial and hematopoietic cells induces HSPC mobilization into the peripheral blood without affecting endothelial cells homeostasis. Recent work from our lab further demonstrated that VCAM1 expression on HSCs and leukemic stem cells confers innate immune tolerance ( Nat Cell Biol 2022). While the contribution of HSC and endothelial-derived VCAM1 to homeostasis has been well studied, the role of MSC-derived VCAM1 on HSC and MSC maintenance and multilineage potency remains unknown. Our supporting data suggest that MSCs are the BM's main source of Vcam1 (p<0.0001) and that VCAM1 is critical for the maintenance, survival, and function of MSCs. Using a mouse model in which Vcam1 can be conditionally deleted in BM MSCs by a tamoxifen-inducible N-Cadherin-Cre ER line (referred to as Vcam1 N-CadCreER), we found that deletion of Vcam1 significantly reduces the number (4-fold, p=0.0030), frequency (4-fold, p=0.0042), and viability (2-fold, p=0.0039) of BM MSCs. Previous findings in breast cancer revealed that VCAM1 promotes PI3K/Akt survival signaling via Akt phosphorylation. Accordingly, Vcam1 deletion in N-Cadherin-Cre ER MSCs, demonstrated a significant reduction in phospho-Akt (Ser473) (p<0.05), supporting the role of VCAM1 in mediating PI3K/Akt survival signaling in MSCs. Intriguingly, the remaining viable VCAM1-null MSCs isolated from Vcam1 N-CadCreER mice exhibited reduced clonogenic potential (determined by CFU-F assays; 3-fold, p=0.0001). Furthermore, micro-CT analysis of 6-month-old Vcam1 N-CadCreER mice presented an altered bone phenotype compared to the control as evidenced by their significantly increased bone thickness and volume (p<0.05), yet significantly decreased tissue mineral density and medullary cavity area (p<0.05). Bulk RNA sequencing (RNAseq) of sorted viable MSCs revealed distinct grouping between Control and Vcam1 N-CadCreER MSCs. Gene ontology analysis demonstrated that deletion of Vcam1 caused an upregulation of pathways associated with inflammation and cytokine release while pathways associated with ossification, extracellular matrix organization, and skeletal development were all downregulated. Surprisingly, both bulk RNAseq and qPCR analysis confirmed a significant reduction in the expression of HSC niche factors ( Vcam1 Cxcl12 and Scf) following Vcam1 deletion, indicative of reduced niche activity. As expected, Vcam1 deletion in N-Cadherin-Cre ER MSCs, caused significant mobilization of HSCs into the blood (p<0.0001) and BM homing defects (p<0.05), however the number and repopulation capacity of BM HSCs remained unaltered, as seen by BM transplantation experiments. Surprisingly, imaging analysis of whole-mount bone marrow sternum of phenotypic HSCs revealed no differences in HSC niche localization between Vcam1-null and Control mice. Accordingly, ELISA analysis of BM extracellular fluid revealed no differences in overall CXCL12 and SCF levels suggesting that there may be some local or systemic niche compensation at play. Single-cell (sc)RNAseq of pooled BM and compact bone sorted non-hematopoietic stromal cells (DAPI -Lineage -CD45 -Ter119 -CD19 -CD71 -)from Vcam1 N-CadCreERand Control mice further confirmed the downregulation of HSC niche factors in MSCs, and overall remodeling of the BM stromal cell composition following Vcam1 deletion. Interestingly, our scRNAseq analysis revealed increased expression of Cxcl12 and Scf in specific vascular and stromal cell populations, supporting the notion of HSC niche compensation. Overall, these findings demonstrate that MSC-derived Vcam1 is critical for BM MSC survival and their HSC niche function.

Neuropeptide Y Attenuates Aging-Induced Bone Marrow Niche Defects and Rejuvenate Aged Hematopoietic Stem/Progenitor Cells

Aging-associated defects in hematopoiesis are linked to declining in hematopoietic stem cell (HSC) self-renewal and multilineage differentiation capacity. The bone marrow (BM) microenvironment/niche has been suggested to influence HSC aging. However, the factors/mechanisms whereby the BM niche mediates hematopoietic stem and progenitor (HSPC) aging are poorly understood. We recently discovered that neuropeptide Y (NPY) plays a crucial role in HSPC trafficking by regulating the BM vascular gateway function (PMCID: PMC5707149). NPY is one of the most abundant neurotransmitters released in the BM by sympathetic nerve fibers. In this study, we found that the aging-induced deficit of NPY levels in the BM plays a critical role in niche defects and HSPC aging by impairing the molecular pathways involved in regulating oxidative stress, senescence, and stemness. The measurement of NPY levels in the BM of young (3-4 months) and aged (18-23 months) mice showed lower NPY levels in aged mice than in young mice (2.2-fold), which was associated with a decrease in mesenchymal stem cells (MSC) (2-fold, p=0.008) and endothelial cells (EC) (1.5-fold, p<0.05) numbers in the BM. In addition, we found that circulating NPY levels decline in the older human population (65-83 years) than in young/ middle age (23-55 years) individuals (1.5-fold, p=0.01). We hypothesized if aging-induced NPY deficit contributes to BM niche defects and HSPC aging, then NPY supplementation may attenuate/revert these processes. To explore this possibility, we administered exogenous NPY in aged mice (100ng/mouse) for 15 consecutive days and evaluated HSPC and niche cells. NPY supplementation in aged mice (18-23 months) substantially reverted aged HSPC phenotype, including NPY decreased the accumulation of HSC-enriched SLAM LSK (Lin-Sca-1+c-Kit+CD48-CD150+) in the BM (1.8-fold, p<0.05) and increased the HPC clonal expansion (1.9-fold, p<0.05) measured by flow cytometry and CFU-C assay, respectively. Competitive BM transplantation studies using donor cells from young, aged, or NPY-treated aged mice into lethally irradiated syngeneic mice showed myeloid biased repopulation from aged mice donor cells than young mice donor cells, and that was attenuated in NPY-treated aged mice donor cells (33%, p=0.003). In addition, NPY substantially reverted aging-related reduction in BM MSC (CD45-Ter119-CD31-Nestin+PDGFR+CD51+) and EC (CD45-Ter119-CD31+VE-cadherin+) numbers, and BM MSC clonal expansion (2-fold, p<0.05) as measured by CFU-F assay. To further validate NPY's role in the aged BM niche and HSPC function, we used NPY gene overexpressing and knockout (KO) mouse models. Compared to wild-type (WT) aged mice, NPY overexpressing aged mice showed a substantial increase in BM niche MSC numbers (1.4-fold, P=0.03) and improved HSPC clonal expansion (2.5-fold, P=0.002). In contrast, 16-18 months old NPY KO mice showed enhanced aging than aged WT mice, reflected by fewer MSC counts and lower HSPC clonal expansion (MSC: 1.6-fold, and CFU-F: 1.5-fold, <0.05). Oxidative stress and senescence are the hallmarks of aging. To examine if NPY attenuates HSPC aging by controlling oxidative and senescence, we measured reactive oxygen species (ROS) and senescence in SLAM-LSK of young, aged, and NPY-treated aged mice using CellROX and anti-p16 antibodies, respectively. The SLAM-LSK from aged mice showed higher expression of ROS (9-fold, p=0.03) and p16 than young mice (1.7-fold). However, ROS and p16 expression were decreased in NPY-treated aged mice (ROS: 2-fold, and p16: 1.3-fold, p<0.05). To identify the molecular pathways involved in NPY-mediated attenuation of HSPC aging, we performed RNA sequencing analysis of SLAM-LSK cells. Aged mice SLAM LSK showed impaired expression of genes involved in pathways regulating antioxidant defense, DNA damage repair and stemness than in young mice SLAM LSK. However, NPY treatment in aged mice substantially restored these pathways in SLAM LSK. In conclusion, our studies suggest that an aging-induced deficit in NPY levels in the BM is a crucial lesion contributing to niche defects and HSPC aging. Thus, NPY supplementation or protection of NPY-producing nerve fibers in the BM can attenuate/restore aging-induced niche damage and rejuvenate aged HSPC.

Pre-Leukemic Transformation of the Bone Marrow Microenvironment Induced By AML1-ETO Fusion Protein

Objective: The bone marrow (BM) microenvironment, especially stromal cells (SCs) and cytokines, plays a critical role in supporting normal hematopoiesis. However, in cases of hematological malignancies such as acute myeloid leukemia (AML), leukemia cells can reshape the BM microenvironment to promote their survival. Despite this, there is still limited understanding of the changes in AML1-ETO fusion protein induced pre-leukemia microenvironment niche. In this study, the alterations of the BM microenvironment in the Aml1 Eto/+; Mx1-Cre (AE KI) mouse model are thoroughly explored and analyzed. This investigation will encompass the study of SCs, cytokines, their sources, and their potential impacts on the occurrence and progression of AML induced by AML1-ETO. Methods: After 4 weeks of PIPC induction, proportion changes of endothelial cells (ECs), osteoblasts, mesenchymal stem cells (MSCs) in the BM of Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group) were analyzed by flow cytometry. Bulk RNA sequencing (RNAseq) and single cell RNA sequencing (scRNAseq) techniques were applied for analysis of abnormal hematopoietic Lin -Sca-1 +c-Kit + (LSK) stem cells. Cytokine levels were detected using ELISA and Luminex bead assay. Additionally, CRISPR-Cas9 technology was used to knock out the target genes and their functions were validated. Results: Following 4 weeks of PIPC induction, the experimental group exhibited a considerable reduction in the proportion and absolute number of ECs, osteoblasts and MSCs, with ECs being the most significantly reduced. The Luminex assay revealed a substantial increase in cytokine release in the BM of the experimental group, including IL4, IL10, TNFa, Cxcl10, Cxcl11, Ccl1, Ccl2, Ccl4, Ccl7, etc. Bulk RNAseq studies of LSK cells showed that the inflammatory pathway in the experimental group was significantly upregulated. To further explore the release sources of cytokines in the BM and heterogeneity in LSK cell cytokine secretion, scRNAseq analysis of LSK cells was conducted in the AE KI group, which were classified into 9 subgroups. Subgroups 1 and 5 exhibited a significant upregulation of Ccl2, Ccl4, Cxcl2, and IL15. Subgroups 4 and 8 showed upregulation of Ccl2 and IL4. Subgroups 0 and 2 exhibited upregulation of Ccl4, while subgroups 6, 7, and 9 showed upregulation of Cxcl2. Notably, subgroups 1 and 5 also displayed increased expression of Ccr5, which is the receptor for Ccl2. To further validate the origins of cytokines, LSK cells were sorted from experimental group and the control group for in vitro culture, respectively. Subsequently, the cell culture supernatants were collected for Luminex assay, revealing a notable upregulation of Ccl2 and Ccl7 in the experimental group. Furthermore, when comparing m-cherry + LSK cells (AML1-ETO positive) with m-cherry - LSK cells (AML1-ETO negative) from the same mouse, a significant increase in the Ccl2 levels in the m-cherry + LSK cell culture supernatant was observed. Flow cytometry analysis of LSK cells further revealed high expression of Ccl2 and its receptor Ccr5 in Ccl2 + LSK cells. In order to gain a deeper understanding of the functions of Ccl2 and Ccl7, CRISPR-Cas9 technology was used to knockout Ccl2 and Ccl7 in c-kit + cells, which were then transplanted into mice for further investigation, respectively. After 3 months, partial restoration of ECs was observed compared to that of the scramble control group. Conclusion: Our findings suggested that significant changes occurred in the BM microenvironment in AML1-ETO-induced pre-leukemia stage. These changes were characterized by a decrease in SCs and an increase in pro-inflammatory cytokine levels. Moreover, our study identified the heterogeneity in cytokine transcriptional profiles among AE KI LSK cells. The secretion of Ccl2 and Ccl7 by AE KI LSK cells with a blockage in hematopoietic differentiation seemed to be involved in the reduction of stromal cell population. These results provided valuable insights for further exploration of the intricate interactions between abnormal hematopoietic stem cells and the BM microenvironment in AML1-ETO-induced pre-leukemic and leukemic states. Understanding these interactions is crucial for advancing our knowledge of the underlying mechanisms involved in the development and progression of AML.

Unlocking the potential of Mesenchymal stem cells in liver Fibrosis: Insights into the impact of autophagy and aging.

Liver fibrosis is a chronic liver disease characterized by extracellular matrix protein accumulation, potentially leading to cirrhosis or hepatocellular carcinoma. Liver cell damage, inflammatory responses, and apoptosis due to various reasons induce liver fibrosis. Although several treatments, such as antiviral drugs and immunosuppressive therapies, are available for liver fibrosis, they only provide limited efficacy. Mesenchymal stem cells (MSCs) have become a promising therapeutic option for liver fibrosis, because they can modulate the immune response, promote liver regeneration, and inhibit the activation of hepatic stellate cells that contribute to disease development. Recent studies have suggested that the mechanisms through which MSCs gain their antifibrotic properties involve autophagy and senescence. Autophagy, a vital cellular self-degradation process, is critical for maintaining homeostasis and protecting against nutritional, metabolic, and infection-mediated stress. The therapeutic effects of MSCs depend on appropriate autophagy levels, which can improve the fibrotic process. Nonetheless, aging-related autophagic damage is associated with a decline in MSC number and function, which play a crucial role in liver fibrosis development. This review summarizes the recent advancements in the understanding of autophagy and senescence in MSC-based liver fibrosis treatment, presenting the key findings from relevant studies.

Investigating the Interplay Between Matrix Compliance and Passaging History on Chondrogenic Differentiation of Mesenchymal Stem Cells Encapsulated Within Alginate-Gelatin Hybrid Hydrogels

Mesenchymal stem cells (MSCs) are used widely in tissue engineering and regenerative medicine because of their ease of isolation and their pluripotency. The low survival and retention rate of MSCs at the target site upon implantation can be addressed via encapsulation within hydrogels capable of directing their fate. In this study, the interplay between matrix mechanics and the passage number of MSCs on their chondrogenic differentiation was assessed. Human bone marrow-derived MSCs between passages 4 and 6 were encapsulated within alginate-gelatin hybrid gels. The stiffness of the gels was varied by varying alginate concentrations while maintaining the concentration of gelatin and consequently, the cell adhesion sites, constant. The study revealed that within 4.8 kPa gels, GAG deposition was higher by P4 MSCs compared to P6 MSCs. However, an opposite trend was observed with collagen type 2 deposition. Further, we observed enhanced chondrogenic differentiation upon encapsulation of MSCs within 6.7 kPa hydrogel irrespective of passaging history. However, the effect of matrix compliance was more prominent in the case of higher passaged MSCs suggesting that matrix stiffness can help rescue the reduced differentiation capability of these cells.

Thymidine Phosphorylase Deficiency or Inhibition Preserves Cardiac Function in Mice With Acute Myocardial Infarction.

Background Ischemic cardiovascular disease is the leading cause of death worldwide. Current pharmacologic therapy has multiple limitations, and patients remain symptomatic despite maximal medical therapies. Deficiency or inhibition of thymidine phosphorylase (TYMP) in mice reduces thrombosis, suggesting that TYMP could be a novel therapeutic target for patients with acute myocardial infarction (AMI). Methods and Results A mouse AMI model was established by ligation of the left anterior descending coronary artery in C57BL/6J wild-type and TYMP-deficient (Tymp-/-) mice. Cardiac function was monitored by echocardiography or Langendorff assay. TYMP-deficient hearts had lower baseline contractility. However, cardiac function, systolic left ventricle anterior wall thickness, and diastolic wall strain were significantly greater 4 weeks after AMI compared with wild-type hearts. TYMP deficiency reduced microthrombus formation after AMI. TYMP deficiency did not affect angiogenesis in either normal or infarcted myocardium but increased arteriogenesis post-AMI. TYMP deficiency enhanced the mobilization of bone marrow stem cells and promoted mesenchymal stem cell (MSC) proliferation, migration, and resistance to inflammation and hypoxia. TYMP deficiency increased the number of larger MSCs and decreased matrix metalloproteinase-2 expression, resulting in a high homing capability. TYMP deficiency induced constitutive AKT phosphorylation in MSCs but reduced expression of genes associated with retinoid-interferon-induced mortality-19, a molecule that enhances cell death. Inhibition of TYMP with its selective inhibitor, tipiracil, phenocopied TYMP deficiency, improved post-AMI cardiac function and systolic left ventricle anterior wall thickness, attenuated diastolic stiffness, and reduced infarct size. Conclusions This study demonstrated that TYMP plays an adverse role after AMI. Targeting TYMP may be a novel therapy for patients with AMI.

Evaluation of Expansion and Maintenance of Umbilical Cord Blood CD34+ Cells in The Co-Culture with Umbilical Cord Blood-Derived Mesenchymal Stem Cells in The Presence of Microcarrier Beads.

Umbilical cord blood (UCB) is an accessible and effective alternative source for hematopoietic stem cell (HSC) transplantation. Although the clinical application of UCB transplantation has been increased recently, quantitative limitation of HSCs within a single cord blood unit still remains a major hurdle for UCB transplantation. In this study we used microcarrier beads to evaluate the ex vivo expansion of UCB-derived HSCs in co-cultured with UCB-derived mesenchymal stem cells (MSC). In this experimental study, we used microcarrier beads to expand UCB-derived MSCs. We investigated the simultaneous co-culture of UCB-derived CD34+ cells and MSCs with microcarrier beads to expand CD34+ cells. The colony forming capacity and stemness-related gene expression on the expanded CD34+ cells were assessed to determine the multipotency and self-renewal of expanded cells. Our results indicated that the microcarrier-based culture significantly increased the total number and viability of UCB-derived MSCs in comparison with the monolayer cultures during seven days. There was a significant increase in the UCB-derived CD34+ cells expanded in the presence of microcarrier beads in this co-culture system. The expanded UCB-derived CD34+ cells had improved clonogenic capacity, as evidenced by higher numbers of total colony counts, granulocyte, erythrocyte, monocyte, megakaryocyte colony forming units (CFU-GEMM), and granulocyte-monocyte colony forming units (CFU-GM). There were significantly increased expression levels of key regulatory genes (CXCR4, HOXB4, BMI1) during CD34+ cells self-renewal and quiescence in the microcarrier-based co-culture. Our results showed that the increase in the expansion and multipotency of CD34+ cells in the microcarrierbased co-culture can be attributed to the enhanced hematopoietic support of UCB-derived MSCs and improved cell-cell interactions. It seems that this co-culture system could have the potential to expand primitive CD34+ cells.

Dynamic MRI of the Mesenchymal Stem Cells Distribution during Intravenous Transplantation in a Rat Model of Ischemic Stroke.

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell-cell interactions or invoking direct long-lasting effects on brain vessels.

Aqueous extract of Achillea millefolium significantly affects mesenchymal stem cell renewal and differentiation in a dose dependent manner.

Achillea millefolium is an Iranian herbal medicine with various effects on the human cells. The aim of this study was to investigate the effects of the aqueous extract of Achillea millefolium (AEAM) on the proliferation and differentiation of mesenchymal stem cells (MSCs). In this study, bone marrow-MSCs (BM-MSCs) were obtained from Wister rat bone morrow and then cultured in Dulbecco's modified Eagle's medium /Nutrient Ham's Mixture F-12 (DMEM/F12) media. Then, the isolated MSCs were cultured in either osteocyte or adipocyte differentiation media containing 0.2 or 2 mg/mL AEAM and assessed using specific staining method. The isolated BM-MSCs exhibited fibroblast-like morphology and were positive for CD73, and CD90, while negative for CD34 and CD45. AEAM significantly increased self-renewal of BM-MSCs at low dose (0.2 mg/ml, P= 0.001) and increased the pool stem cells in both osteocyte and adipocyte differentiation media. AEAM at low doses may be used in cases where there is a need for large number of stem cells, via increased numbers of MSCs, and help tissue repair and immunomodulation.

Bead-jet printing enabled sparse mesenchymal stem cell patterning augments skeletal muscle and hair follicle regeneration

Transplantation of mesenchymal stem cells (MSCs) holds promise to repair severe traumatic injuries. However, current transplantation practices limit the potential of this technique, either by losing the viable MSCs or reducing the performance of resident MSCs. Herein, we design a “bead-jet” printer, specialized for high-throughput intra-operative formulation and printing of MSCs-laden Matrigel beads. We show that high-density encapsulation of MSCs in Matrigel beads is able to augment MSC function, increasing MSC proliferation, migration, and extracellular vesicle production, compared with low-density bead or high-density bulk encapsulation of the equivalent number of MSCs. We find that the high-density MSCs-laden beads in sparse patterns demonstrate significantly improved therapeutic performance, by regenerating skeletal muscles approaching native-like cell density with reduced fibrosis, and regenerating skin with hair follicle growth and increased dermis thickness. MSC proliferation within 1-week post-transplantation and differentiation at 3 − 4 weeks post-transplantation are suggested to contribute therapy augmentation. We expect this “bead-jet” printing system to strengthen the potential of MSC transplantation.

Mesenchymal Stem Cell Identification After Delayed Cord Clamping.

We sought to determine the feasibility of identifying and quantifying mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) after delayed cord clamping in preterm and term births. We obtained 3mL of UCB at various gestational ages after delayed cord clamping. UCB separated by density gradient centrifugation within 4h of delivery was passed through magnetic bead micro-columns to exclude the CD34 + cell population. The samples were incubated with fluorescent-tagged mesenchymal cell marker antibodies CD 29, CD44, CD73, CD105, and hematopoietic cell marker CD45. The cell populations were analyzed by flow cytometry. Viable cells were assessed with 7-aminoactinomycin-D. The results were expressed in median (minimum to maximum) MSCs and compared between preterm and term samples. A total of 12 UCB samples (32-40weeks) were obtained, 10 of which demonstrated MSCs, accounting for 0.0174% (0-14.7%) of the viable UCB mononuclear cells. MSCs comprised 0.148% (0.0006-1.59%) and 0.116% (0-14.7%) of the viable UCB mononuclear cells in the term (n = 5), 38.4 ± 1.3weeks, and preterm (n = 7) samples, 34.6 ± 1.1, respectively, p = 0.17. There was an overall median of 96 (0-39,574) MSCs. There was no difference in the median numbers of MSCs identified between term and preterm UCB samples, 3384 (23-6042) and 36 (0-39,574), respectively, p = 0.12. Mesenchymal stem cells were identified and quantified in 5 of 7 preterm and all 5 term UCB 3-mL samples obtained after delayed cord clamping.

A pilot study to demonstrate the paracrine effect of equine, adult allogenic mesenchymal stem cells in vitro, with a potential for healing of experimentally-created, equine thoracic wounds in vivo.

Regenerative biological therapies using mesenchymal stem cells (MSCs) are being studied and used extensively in equine veterinary medicine. One of the important properties of MSCs is the cells' reparative effect, which is brought about by paracrine signaling, which results in the release of biologically active molecules, which in turn, can affect cellular migration and proliferation, thus a huge potential in wound healing. The objective of the current study was to demonstrate the in vitro and in vivo potentials of equine allogenic bone marrow-derived MSCs for wound healing. Equine bone marrow-derived MSCs from one allogenic donor horse were used. Equine MSCs were previously characterized for their in vitro proliferation, expression of cluster-of-differentiation markers, and trilineage differentiation. MSCs were first evaluated for their migration using an in vitro wound healing scratch assay, and subsequently, the conditioned medium was evaluated for their effect on human fibroblast proliferation. Subsequently, allogenic cells were intradermally injected into full-thickness, cutaneous thoracic wounds of 4 horses. Wound healing was assessed by using 3-D digital imaging and by measuring mRNA expression of pro-and anti-inflammatory markers for 30 days. Using human fibroblasts in an in vitro wound healing assay, we demonstrate a significantly higher healing in the presence of conditioned medium collected from proliferating MSCs than in the presence of medium containing fetal bovine serum. The in vitro effect of MSCs did not translate into a detectable effect in vivo. Nonetheless, we proved that molecularly characterized equine allogenic MSCs do not illicit an immunologic response. Investigations using MSCs derived from other sources (adipose tissue, umbilical cord), or a higher number of MSCs or a compromised animal model may be required to prove the efficacy of equine MSCs in wound healing in vivo.

Platelet-Rich Plasma Lysate-Incorporating Gelatin Hydrogel as a Scaffold for Bone Reconstruction.

In implant dentistry, large vertical and horizontal alveolar ridge deficiencies in mandibular and maxillary bone are challenges that clinicians continue to face. One of the limitations of porous blocks for reconstruction of bone in large defects in the oral cavity, and in the musculoskeletal system, is that fibrin clot does not adequately fill the interior pores and does not persist long enough to accommodate cell migration into the center of the block. The objective of our work was to develop a gelatin-based gel incorporating platelet-rich plasma (PRP) lysate, to mimic the role that a blood clot would normally play to attract and accommodate the migration of host osteoprogenitor and endothelial cells into the scaffold, thereby facilitating bone reconstruction. A conjugate of gelatin (Gtn) and hydroxyphenyl propionic acid (HPA), an amino-acid-like molecule, was commended for this application because of its ability to undergo enzyme-mediated covalent cross-linking to form a hydrogel in vivo, after being injected as a liquid. The initiation and propagation of cross-linking were under the control of horseradish peroxidase and hydrogen peroxide, respectively. The objectives of this in vitro study were directed toward evaluating: (1) the migration of rat mesenchymal stem cells (MSCs) into Gtn–HPA gel under the influence of rat PRP lysate or recombinant platelet-derived growth factor (PDGF)-BB incorporated into the gel; (2) the differentiation of MSCs, incorporated into the gel, into osteogenic cells under the influence of PRP lysate and PDGF-BB; and (3) the release kinetics of PDGF-BB from gels incorporating two formulations of PRP lysate and recombinant PDGF-BB. Results: The number of MSCs migrating into the hydrogel was significantly (3-fold) higher in the hydrogel group incorporating PRP lysate compared to the PDGF-BB and the blank gel control groups. For the differentiation/osteogenesis assay, the osteocalcin-positive cell area percentage was significantly higher in both the gel/PRP and gel/PDGF-BB groups, compared to the two control groups: cells in the blank gels grown in cell expansion medium and in osteogenic medium. Results of the ELISA release assay indicated that Gtn–HPA acted as an effective delivery vehicle for the sustained release of PDGF-BB from two different PRP lysate batches, with about 60% of the original PDGF-BB amount in the two groups remaining in the gel at 28 days. Conclusions: Gtn–HPA accommodates MSC migration. PRP-lysate-incorporating hydrogels chemoattract increased MSC migration into the Gtn–HPA compared to the blank gel. PRP-lysate- and the PDGF-BB-incorporating gels stimulate osteogenic differentiation of the MSCs. The release of the growth factors from Gtn–HPA containing PRP lysate can extend over the period of time (weeks) necessary for bone reconstruction. The findings demonstrate that Gtn–HPA can serve as both a scaffold for cell migration and a delivery vehicle that allows sustained and controlled release of the incorporated therapeutic agent over extended periods of time. These findings commend Gtn–HPA incorporating PRP lysate for infusion into porous calcium phosphate blocks for vertical and horizontal ridge reconstruction, and for other musculoskeletal applications.