scholarly journals Adipose Stem Cell-Derived Exosomes As a Macrophage-Based Treatment for Chronic Rotator Cuff Tendinopathy: From a Mouse Model to a Study in Human Tissue

2021 ◽  
Vol 9 (7_suppl4) ◽  
pp. 2325967121S0020
Author(s):  
Weilin Yu ◽  
wei song ◽  
Xudong Liu ◽  
Yaohua He ◽  
Chongyang Wang
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Rotator Cuff ◽  
Human Tissue ◽  
Saline Group ◽  
In Vivo Model ◽  
Ground Substance ◽  
Mouse Bone Marrow ◽  
Adipose Stem Cell ◽  
Rotator Cuff Tendinopathy

Objectives: Chronic rotator cuff tendinopathy is one of the most prevalent causes of shoulder pain. Growing evidence suggests that macrophages play a significant role in the pathogenesis of tendinopathy. Therefore, a treatment that targets macrophages would be useful for patients with this common musculoskeletal disorder. To investigate whether adipose stem cell-derived exosomes (ASC-Exos) mediate polarization of macrophages and contribute to the healing of chronic rotator cuff tendinopathy. Methods: First, we compared the effects of ASC-Exos on polarization of mouse bone marrow-derived macrophages between a classically activated phenotype (M1φ) and an alternatively activated phenotype (M2φ) in vitro. Seventy-two C57Bl/6 mice were assigned to normal cage activity (n=24) or 5 weeks of treadmill overuse (n=48). The enthesis of supraspinatus in each mouse was then injected with ASC-Exos (ASC-Exos group, n=24) or saline (saline group, n=24). Histological and biomechanical outcomes were assessed 4 weeks after treatment. Finally, tissue samples from human patients with rotator cuff tendinopathy were obtained to determine the effect of ASC-Exos on the M1φ/M2φ balance in tissue-resident macrophages. Results: ASC-Exos inhibited M1φ polarization and augmented M2φ polarization both in mouse bone marrow-derived macrophages, mouse in vivo model and human tissue-resident macrophages. Mice in the ASC-Exos group showed less severe pathological changes than the saline group, including less cellular infiltration, disorganization of collagen, and ground substance deposition. The mean modified Bonar score was significantly lower in the ASC-Exos group than in the saline group (9.81 ± 0.96 vs 7.69 ± 1.03; P<.05). Furthermore, the maximum failure load was significantly higher in the ASC-Exos group than in the saline group (4.23 ± 0.66 N vs 3.86 ± 0.65 N; P<.05) as was stiffness (3.38 ± 0.34 N/m vs. 2.68 ± 0.49 N/m; P<.05). Conclusions: ASC-Exos-mediated balance of M1φ/M2φ contributes to healing of tendinopathy. Regulation of the M1φ/M2φ balance could be a new target for treatment of chronic rotator cuff tendinopathy.

Adipose Stem Cell–Derived Exosomes Ameliorate Chronic Rotator Cuff Tendinopathy by Regulating Macrophage Polarization: From a Mouse Model to a Study in Human Tissue

2021 ◽  
Vol 49 (9) ◽  
pp. 2321-2331
Author(s):  
Chongyang Wang ◽  
Yao Zhang ◽  
Guangcheng Zhang ◽  
Weilin Yu ◽  
Yaohua He
Keyword(s):  
Stem Cell ◽  
Rotator Cuff ◽  
Macrophage Polarization ◽  
Saline Group ◽  
Ground Substance ◽  
M2 Macrophage ◽  
Mouse Bone Marrow ◽  
Adipose Stem Cell ◽  
Proinflammatory Response

Background: Chronic rotator cuff (RC) tendinopathy is one of the most prevalent causes of shoulder pain. Growing evidence suggests that macrophages play a significant role in the proinflammatory response, resolution of inflammation, and tissue healing of tendinopathy. In particular, enhancement of M2 macrophage (M2φ) activity contributes to the accelerated healing of tendinopathy. Therefore, a treatment that enhances M2φ polarization would be useful for patients with this common musculoskeletal disorder. Purpose: To investigate whether adipose stem cell–derived exosomes (ASC-Exos) enhance M2φ polarization and ameliorate chronic RC tendinopathy. Study Design: Controlled laboratory study. Methods: First, we compared the effects of ASC-Exos on polarization of mouse bone marrow–derived macrophages between a classically activated phenotype (M1φ) and an alternatively activated phenotype (M2φ) in vitro. In total, 72 C57BL/6 mice were assigned to normal cage activity (n = 24) or 5 weeks of treadmill overuse (n = 48). The supraspinatus tendon of each treadmill overuse mouse was treated with ASC-Exos (n = 24) or saline (n = 24). Histological and biomechanical outcomes were assessed 4 weeks after treatment. Finally, tissue samples from human patients with RC tendinopathy were obtained to assay the effect of ASC-Exos on the M1φ/M2φ balance in tissue-resident macrophages. Results: ASC-Exos inhibited M1φ polarization and augmented M2φ polarization in vitro and in vivo. Mice in the ASC-Exos group showed less severe pathological changes than those in the saline group, including less cellular infiltration, disorganization of collagen, and ground substance deposition. The modified Bonar score of the ASC-Exos group (mean ± SD, 7.68 ± 1.03) was significantly lower than that of the saline group (9.81 ± 0.96; P < .05). Furthermore, the maximum failure load was significantly higher in the ASC-Exos group than in the saline group (4.23 ± 0.66 N vs 3.86 ± 0.65 N; P < .05), as was stiffness (3.38 ± 0.34 N/m vs 2.68 ± 0.49 N/m; P < .05). Conclusion: ASC-Exos–mediated polarization balance of M1φ/M2φ contributes to the amelioration of chronic RC tendinopathy. Regulation of the M1φ/M2φ balance could be a new target for the treatment of chronic RC tendinopathy. Clinical Relevance: Administration of ASC-Exos is a cell-free approach that may become a novel treatment option for chronic RC tendinopathy and should be explored further.

Adipose Stem Cell–Derived Exosomes Decrease Fatty Infiltration and Enhance Rotator Cuff Healing in a Rabbit Model of Chronic Tears

2020 ◽  
Vol 48 (6) ◽  
pp. 1456-1464 ◽  
Author(s):  
Chongyang Wang ◽  
Qingxiang Hu ◽  
Wei Song ◽  
Weilin Yu ◽  
Yaohua He
Keyword(s):  
Stem Cell ◽  
Rotator Cuff ◽  
Bone Healing ◽  
Fatty Infiltration ◽  
Biomechanical Testing ◽  
Rabbit Model ◽  
Saline Group ◽  
Adipose Stem Cell ◽  
Sham Surgery ◽  
Cuff Repair

Background: Fatty infiltration and poor tendon-bone healing in chronic rotator cuff tears (RCTs) are associated with unsatisfactory prognosis. Adipose stem cell–derived exosomes (ASC-Exos), having multiple biological effects, can prevent muscle degeneration in acute RCTs. However, the effects of ASC-Exos on fatty infiltration and tendon-bone healing in chronic RCTs remain unknown. Purpose: To study the effects of ASC-Exos on fatty infiltration and tendon-bone healing in a chronic RCT rabbit model. Study Design: Controlled laboratory study. Methods: At week 0, we randomly allocated 35 rabbits to receive sham surgery (14 rabbits) or establish a bilateral RCT model (21 rabbits, detachment of the supraspinatus tendon). At week 6, a total of 7 rabbits received sham surgery, and 7 rabbits with RCT were sacrificed for fatty infiltration assay. The remaining 14 rabbits with bilateral RCTs were randomly assigned to a saline group (7 rabbits that received local saline injection and rotator cuff repair) or an ASC-Exos group (7 rabbits that received local ASC-Exos injection and rotator cuff repair). At week 18, all rabbits were sacrificed for histological examination and biomechanical testing. Results: At week 18, the ASC-Exos group showed significantly lower fatty infiltration (14.01% ± 2.85%) compared with the saline group (21.79% ± 3.07%) ( P < .001), and no statistical difference compared with the time of repair (10.88% ± 2.64%) ( P = .127). For tendon-bone healing, the ASC-Exos group showed a higher histological score and more newly regenerated fibrocartilage at the repair site than did the saline group. Regarding biomechanical testing, the ASC-Exos group showed significantly higher ultimate load to failure, stiffness, and stress than the saline group. Conclusion: Local injection of ASC-Exos in chronic RCTs at the time of repair could prevent the progress of fatty infiltration, promote tendon-bone healing, and improve biomechanical properties. Clinical Relevance: ASC-Exos injection may be used as a cell-free adjunctive therapy to inhibit fatty infiltration and improve rotator cuff healing in the repair of chronic RCTs.

scholarly journals Improved techniques for liquid culture of human and mouse bone marrow

Blood ◽  
1976 ◽  
Vol 47 (3) ◽  
pp. 369-379
Author(s):  
MJ Cline ◽  
DW Golde
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Large Scale ◽  
Membrane Surface ◽  
Diffusion Chamber ◽  
Mononuclear Phagocytes ◽  
Mouse Bone Marrow ◽  
Improved Performance ◽  
Proliferation In Vitro

Previous studies using the in vitro diffusion chamber (Marbrook) have shown that bone marrow grown in this system will undergo limited stem cell replication and differentiation to mature granulocytes and mononuclear phagocytes. A series of studies with modified culture systems was initiated to improve cell production and committed stem cell (CFU-C) proliferation in vitro. Introduction of a continuous-flow system and a migration technique providing means of egress for mature neutrophils resulted in substantially improved performance. CFU-C were found to be capable of migration through a 3-mu pore membrane. These studies indicated that membrane surface area, culture medium circulation, and mature cell egress were among the conditions that could be optimized for maximum hematopoietic cell proliferation in suspension culture. The present observations also suggested that large- scale in vitro growth of mammalian bone marrow may be feasible.

scholarly journals Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3932-3939 ◽  
Author(s):  
Yang Du ◽  
Nancy A. Jenkins ◽  
Neal G. Copeland
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Drug Targets ◽  
Insertional Mutagenesis ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Serial Passage ◽  
Mouse Bone Marrow ◽  
Cancer Genes ◽  
Differentiation Potential

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.

Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2846-2846
Author(s):  
M. Kozik ◽  
J. Banks ◽  
L. Fanning ◽  
M. Finney ◽  
Y. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Flow Analysis ◽  
Scid Mice ◽  
Mouse Bone Marrow ◽  
Short Term ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

Dipeptidyl Peptidase 4 (DPP4) Is Altered By, and Modulates Sensitivity to, Extra-Physiologic Oxygen Shock Stress (EPHOSS): Implications for Hematopoietic Stem Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2361-2361
Author(s):  
Heather A. O'Leary ◽  
Thomas McNamara ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ambient Air ◽  
Mouse Bone Marrow ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Dipeptidyl Peptidase 4 ◽  
Expression Activity ◽  
Shock Stress

Abstract Hematopoietic stem cells (HSC) reside in hypoxic niches (~1-4% O2), however, HSC studies are consistently performed using cells isolated in ambient air (~20% O2), regardless of subsequent processing in low oxygen tension. We recently published that by collecting/processing stem cells in physiologically native conditions of hypoxia, with all procedures performed inside a hypoxic chamber (3% O2), we enhance the recovery of phenotypic, and functional, self-renewing long-term repopulating HSC (LT-HSC) with concomitantly decreasing numbers of progenitor cells. This occurs by inhibiting damage due to brief exposure of mouse bone marrow (BM) or human cord blood (CB) cells to ambient oxygen (a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS)) which we, in part, mechanistically linked to mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. This data suggests that true numbers of HSCs, and the transplantation potency of BM and CB, have been consistently underestimated due to rapid differentiation of LT-HSCs in ambient air (EPHOSS), but the broad effects of EPHOSS on stem cell phenotype are unknown. We hypothesized that Dipeptidyl Peptidase 4 (DPP4) may be altered by EPHOSS and involved in the effects of EPHOSS on HSC. We showed that DPP4, a serine peptidase whose enzymatic activity leads to the N terminal cleavage of select penultimate amino acids of proteins, alters homing and engraftment of HSC and the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, the ability of DPP4 T proteins to induce signaling that FL factors cannot, and the effects of EPHOSS on DPP4 expression/activity, and vice versa, have not been investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating that mouse bone marrow harvested in air in the presence of Diprotin A, a DPP4 inhibitor, or from DPP4 K/O mice, results in a significant increase in the number of phenotypic LT-HSC (p=.017), suggesting that inhibition of DPP4 can diminish the loss of phenotypic LT-HSC due to EPHOSS. Further, the percentage of DPP4+ cells is significantly increased in primitive fractions of mouse bone marrow and human cord blood (LSK ~15% DPP4+, LSKCD150+ ~40%DPP4+, CD34+CD38- of CB ~10% DPP4+, CD34+CD38-CD45RA-CD90+CD49F+ ~40% DPP4+, p=.007), the numbers of DPP4+ cells are additionally enhanced 15- 20% when cells (BM and hCB) are isolated in hypoxia, especially in the LT-HSC fraction (Air 40% DPP4+ Hypoxia 60% DPP4+, p=.005). However, DPP4 activity on lineage- bone marrow harvested in hypoxia showed a 2 fold decrease (p=.005) compared to lineage- cells harvested in air. Interestingly, this increase in the number of DPP4+ cells in hypoxia is not recapitulated when mouse BM is harvested in the presence a Cyclosporin A, a cylophilin D inhibitor, (even though the increase in numbers of LT-HSC is preserved similarly to that in hypoxia) suggesting an alternative mechanism for modulation of DPP4 other than inhibition of mitochondrial ROS/MPTP. Unexpectedly, LT-HSC ROS levels (both mitochondrial and total) were not diminished in groups with decreased DPP4 activity (DPA or DPP4 K/O) harvested in air despite the blunting of EPHOSS leading to maintenance of the phenotypic LT-HSC increase over air harvest alone. These data suggest that pathways in addition to ROS, such as DPP4 expression/activity, may be influencing LT-HSC function after, and sensitivity to, EPHOSS as well as being modulated by EPHOSS. Further investigation of these collaborative pathways may facilitate increased HSC collections to enhance HSC transplantation. Disclosures No relevant conflicts of interest to declare.

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