Injection Of Conditioned Medium Research Articles

Conditioned Medium of Infrapatellar Fat Stem Cells Alleviates Degradation of Chondrocyte Extracellular Matrix and Delays Development of Osteoarthritis

Introduction: Osteoarthritis (OA) is a prevalent clinical chronic degenerative condition characterized by the degeneration of articular cartilage. Currently, drug treatments for OA come with varying degrees of side effects, making the development of new therapeutic approaches for OA imperative. Mesenchymal stem cells (MSCs) are known to mitigate the progression of OA primarily through paracrine effects. The conditioned medium (CM) derived from MSCs encapsulates a variety of paracrine factors secreted by these cells. Methods: In this study, we investigated the effect of the CM of infrapatellar fat pad-derived MSCs (IPFSCs) on OA in vitro and in vivo, as well as and the potential underlying mechanisms. We established three experimental groups: the normal group, the OA group, and the CM intervention group. In vitro experiments, we used methods such as qPCR, Western blot, immunofluorescence, and flow cytometry to detect the impact of CM on OA chondrocytes. In vivo experiments, we evaluated the changes in the knee joints of OA rats after intra-articular injection of CM treatment. Results: The results showed that injection of CM into the knee joint inhibited OA development in a rat model induced by destabilization of the medial meniscus and anterior cruciate ligament transection. The CM increased the deposition of extracellular matrix-related components (type II collagen and Proteoglycan). The activation of PI3K/AKT/NF-κB signaling pathway was induced by IL-1β in chondrocytes, which was finally inhibited by CM-IPFSCs treatment. Conclusion: In summary, IPFSCs-CM may have therapeutic potential for OA.

EFFECTS OF PROGENITOR CELL CONDITIONED MEDIA ON THE AMOUNT OF BRAIN CORTEX NEURONS IN A RAT MODEL OF TRAUMATIC BRAIN INJURY

Aim. The purpose of the study was to examine beneficial effect of conditioned media (CM) of progenitor cells of different origin (neurogenic progenitor cells, or NPCs, and adipose-derived mononuclear cells, or AMCs) as a source of mesenchymal multipotent stromal cells (MMSCs) on brain cortex neurons in rats with traumatic brain injury (TBI). Methods. TBI was reproduced in outbred sexually mature male rats by developing the model of free-falling load (50 g) with damage to the left hemisphere of the brain. The rats were injected 3 times with an interval of every other day intraperitoneally with NPCs CM and AMCs CM (dose 0.8 mg by total protein) that were obtained from cell cultures of fetal rat brain and adult rat adipose tissue. On the 5th day after TBI, the morphologic study of brain tissue was performed. Results. The number of neurons in the cortex of rats on the 5th day after TBI in damaged hemisphere as well as in contralateral hemisphere compared to control group decreased by half. Three i.p. injections of NPCs CM or AMCs CM increased the number of neurons in the cortex in both hemispheres in rats of corresponding groups compared to the rats with TBI without additional treatment. Conclusion. Obtained results indicate that CM obtained from NPCs and AMCs have noticeable neuroprotective effect on the damaged neurons and might be considered as an additional mode to treatment of TBI.

Improvement of Corneal Nerve Regeneration in Diabetic Rats Using Wharton's Jelly-Derived Mesenchymal Stem Cells and their Conditioned Medium.

To investigate the efficacy of Wharton's jelly mesenchymal stem cells (WJSCs) and their conditioned medium (CM) for corneal nerve regeneration in rats with diabetic keratopathy. Streptozotocin (STZ)-induced male diabetic (DM) rats (250-300 g) were divided into four groups (n=7/group): Control, DM, DM with WJSCs (DM+WJ), and DM with CM treatment (DM+CM). DM+WJ and DM+CM group received WJSCs or CM, respectively, topically with eye drops. Corneal sensibility, corneal epithelial layer integrity, histology, expression of GAP-43 and TUBB3 on mRNA level and their immunohistochemical expression were examined after two weeks of treatment. There were changes in corneal sensibility and corneal integrity between normal control and diabetic groups with/without WJSC or CM injection. Total central corneal thickness was significantly higher in DM+CM (249.81 ± 43.85 μm) than in control (174.72 ± 44.12 μm, P=0.004) and DM groups (190.15 ± 9.63 μm, P=0.03). GAP-43 mRNA expression levels of DM+WJ and DM+CM groups were higher compared with DM and control groups. TUBB3 mRNA level was increased after CM (P=0.047), but not after WJSCs treatment (P=1.00). GAP-43 and TUBB3 immunohistochemical expression of nerve fibers along the epithelial layer significantly increased in DM+WJ and DM+CM compared with DM group. Our findings showed that WJSCs and their CM improved corneal nerve regeneration in rats with diabetic keratopathy.

Effect of the conditioned medium of mesenchymal stem cells on the expression levels of P2X4 and P2X7 purinergic receptors in the spinal cord of rats with neuropathic pain.

Recent studies have shown that mesenchymal stem cells (MSCs) and their conditioned medium (CM) have potential therapeutic effects in animal models of neuropathic pain (NP). However, the mechanisms underlying these effects are not fully understood. Because of the leading involvement of purinergic receptors in the pathogenesis of NP, this study aimed to investigate the effect of MSCs-CM on the expression levels of P2X4 and P2X7 receptors in a rat model of NP induced by chronic constriction injury (CCI) of the sciatic nerve. CM was prepared from the rats' bone marrow-derived MSCs culture. After that, NP rats were treated by intraperitoneal injection of CM, or Dulbecco's modified Eagle's medium (DMEM) 1day before and 7 and 11days after CCI surgery. The NP status was assessed in the treated animals using behavioral tests, including mechanical allodynia and thermal hyperalgesia, on days - 1, 3, 6, 9, 12, and 15 of the study. At the end of the study (Day 15), the animals were sacrificed, and the relative gene expression of P2X4 and P2X7 receptors were measured in the spinal cord using quantitative real-time PCR. The results demonstrated that in the CM-treated NP rats, mechanical allodynia and thermal hyperalgesia were significantly reduced compared with the DMEM-treated group. In addition, the expression levels of P2X4 and P2X7 receptors were noticeably prevented in the CM-treated group than the control group. These findings indicate that the antinociceptive effects of CM in the NP rats are partly mediated through preventing the upregulation of P2X4 and P2X7 receptors in the spinal cord.

Effect of Hypoxia Preconditioned Adipose-Derived Mesenchymal Stem Cell Conditioned Medium on Cerulein-Induced Acute Pancreatitis in Mice.

Purpose: Acute pancreatitis (AP) is an inflammatory disorder distinguished by tissue injury and inflammation of the pancreas. Using paracrine potential of mesenchymal stem cells (MSCs) provides a useful clinical approach in treating inflammatory diseases. We investigated the therapeutic effects of adipose-derived MSC conditioned medium (CM) and hypoxia preconditioned adipose-derived MSC conditioned medium (HCM) in cerulein-induced AP in mice. Methods: AP was induced in C57BL/6 mice by intraperitoneal injection of cerulein (75 μg/ kg/h × 7 times). One hour following the last injection of cerulein, mice were treated with intraperitoneal injection of CM and HCM (500 µL/mice/30 min × 3 times). Twelve hours following the treatment, serum levels of amylase and lipase were measured. In addition, pancreas pathological changes, immunohistochemical examinations for evaluation of IL-6 expression and pancreatic myeloperoxidase (MPO) enzyme activity were analyzed. Results: The in vitro results of the morphological, differentiation and immunophenotyping analyses confirmed that hypoxia preconditioned MSCs (HP-MSCs) conserve MSCs characteristics after preconditioning. However, HP-MSCs significantly expressed high mRNA level of hypoxia inducible factor 1-α and higher level of total protein. The in vivo findings of the current study showed that CM and HCM significantly reduced the amylase & lipase activity, the severity of pancreas tissue injury and the expression of IL-6 and MPO enzyme activity compared with the AP group. However, no significant difference between CM and HCM groups was demonstrated. Conclusion: Use of CM and HCM can attenuate cerulein-induced AP and decrease inflammation in the pancreas tissue in AP mice.

Regulation of bone metabolism by megakaryocytes in a paracrine manner

Megakaryocytes (MKs) play key roles in regulating bone metabolism. To test the roles of MK-secreted factors, we investigated whether MK and promegakaryocyte (pro-MK) conditioned media (CM) may affect bone formation and resorption. K562 cell lines were differentiated into mature MKs. Mouse bone marrow macrophages were differentiated into mature osteoclasts, and MC3T3-E1 cells were used for osteoblastic experiments. Bone formation was determined by a calvaria bone formation assay in vivo. Micro-CT analyses were performed in the femurs of ovariectomized female C57B/L6 and Balb/c nude mice after intravenous injections of MK or pro-MK CM. MK CM significantly reduced in vitro bone resorption, largely due to suppressed osteoclastic resorption activity. Compared with pro-MK CM, MK CM suppressed osteoblastic differentiation, but stimulated its proliferation, resulting in stimulation of calvaria bone formation. In ovariectomized mice, treatment with MK CM for 4 weeks significantly increased trabecular bone mass parameters, such as bone volume fraction and trabecular thickness, in nude mice, but not in C57B/L6 mice. In conclusion, MKs may secrete anti-resorptive and anabolic factors that affect bone tissue, providing a novel insight linking MKs and bone cells in a paracrine manner. New therapeutic agents against metabolic bone diseases may be developed from MK-secreted factors.

Conditioned medium obtained from human amniotic membrane-derived mesenchymal stem cell attenuates heart failure injury in rats.

Objective(s):Heart failure (HF) is one of the leading causes of death worldwide. Due to beneficial effects of stem cells, paracrine secretion of them has recently been used by researchers. The purpose of this study was to investigate the effects of intravenous injection (IV) of conditioned medium (CM) of human amniotic membrane-derived mesenchymal stem cell (MSC-CM) on HF.Materials and Methods:Male Wistar rats (n=35, 180 g) were randomly divided into five groups: sham, HF, HF+MSC-CM, HF+culture medium and HF+phosphate-buffered saline (PBS). To induce HF, isoproterenol (170 mg/kg/d) was injected subcutaneously for 4 consecutive days. After 28 days, induction of HF was evaluated by echocardiography. A day after echocardiography, 50 μg culture medium/5 ml PBS in HF+culture medium group, 50 μg MSC-CM/5 ml PBS in HF+MSC-CM group and 5 ml PBS in HF+PBS group were injected two times for 4 successive days. The echocardiography was performed 4 weeks after the last injection of isoproterenol. To evaluate the fibrosis, morphology, and cardiac function, Trichrome Masson’s staining, Hematoxylin and Eosin staining and echocardiography were performed, respectively.Results:CM significantly increased fractional shortening and ejection fraction, and also significantly decreased apoptotic nuclear condensation. Moreover, significant decreased level of fibrosis and increased level of angiogenesis was observed in the treatment group (P<0.05).Conclusion:Our results indicated that IV injection of CM has therapeutic effects on HF by reducing fibrosis and preventing the progression of failure due to its paracrine effects.

Enhancing the Wound Healing Effect of Conditioned Medium Collected from Mesenchymal Stem Cells with High Passage Number Using Bioreducible Nanoparticles.

Injecting human mesenchymal stem cells (hMSCs) at wound sites is known to have a therapeutic effect; however, hMSCs have several limitations, such as low viability and poor engraftment after injection, as well as a potential risk of oncogenesis. The use of a conditioned medium (CM) was suggested as an alternative method for treating various wounds instead of direct hMSC administration. In addition to not having the adverse effects associated with hMSCs, a CM can be easily mass produced and can be stored for long-term, thereby making it useful for clinical applications. In general, a CM is collected from hMSCs with low passage number; whereas, the hMSCs with high passage number are usually discarded because of their low therapeutic efficacy as a result of reduced angiogenic factor secretion. Herein, we used a CM collected from high passage number (passage 12, P12) hMSCs treated with gold-iron nanoparticles (AuFe NPs). Our AuFe NPs were designed to release the iron ion intracellularly via endocytosis. Endosomes with low pH can dissolve iron from AuFe NPs, and thus, the intracellularly released iron ions up-regulate the hypoxia-inducible factor 1α and vascular endothelial growth factor (VEGF) expression. Through this mechanism, AuFe NPs improve the amount of VEGF expression from P12 hMSCs so that it is comparable to the amount of VEGF expression from low passage number (passage 6, P6), without treatment. Furthermore, we injected the CM retrieved from P12 MSCs treated with AuFe NPs in the mouse skin wound model (AuFe P12 group). AuFe P12 group revealed significantly enhanced angiogenesis in the mouse skin wound model compared to the high passage hMSC CM-injected group. Moreover, the result from the AuFe P12 group was similar to that of the low passage hMSC CM-injected group. Both the AuFe P12 group and low passage hMSC CM-injected group presented significantly enhanced re-epithelization, angiogenesis, and tissue remodeling compared to the high passage hMSC CM-injected group. This study reveals a new strategy for tissue regeneration based on CM injection without considering the high cell passage count.

Adipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis.

The immunogenic collagen V (Col V) and the proinflammatory cytokine interleukin (IL)-17 have been implicated in the pathogenesis of multiple autoimmune diseases. Col V is also up-regulated during adipogenesis and can stimulate adipocyte differentiation in vitro. Conditioned medium (CM) generated from adipose-derived mesenchymal stem cells (MSCs) reduces bleomycin (BLM)-induced lung injury in rats, suggesting a crucial role in situ of immunomodulatory factors secreted by MSCs in these beneficial effects. In the present work, we investigated this hypothesis, analyzing levels of plasma inflammatory mediators and inflammatory and fibrotic mediators in the lung tissue of BLM-injured rats after treatment with MSCs and CM. Pulmonary fibrosis was intratracheally induced by BLM. After 10 days, BLM animals were further randomized into subgroups receiving saline, MSCs, or CM intravenously. On days 14 and 21, the animals were euthanized, and the lungs were examined through protein expression of nitric oxide synthase (NOS), IL-17, transforming growth factor-β (TGF-β), vascular endothelial growth factor, endothelin-1, and the immunogenic Col V through histological quantitative evaluation and plasma levels of fibrinogen, Von Willebrand factor, and platelet-derived growth factor (PDGF). Rats that had been injected with MSCs and CM showed a significant increase in weight and significant improvements at 14 and 21 days after intravenous injection at both time points of analysis of plasma fibrinogen, PDGF, and Von Willebrand factor and NOS-2 expression, supporting an early anti-inflammatory action, thus reducing TGF-β and collagen I fibers. In contrast, intravenous injection of CM was able to significantly increase the deposition of Col V fibers and IL-17 on both day 14 and day 21 as compared with the amount observed in rats from the BLM group and MSC groups. In conclusion, this study reinforces previous observations on the therapeutic properties of MSCs and CM and is the first report to demonstrate the association of its actions with immunomodulatory biomarkers on lung tissue. We concluded that adipose-derived stem cells and adipose-derived stem cells-CM modulate an in situ imbalance between collagen I- and Col V-mediated IL-17 immune response, emerging as a promising therapeutic option for recovering from BLM pulmonary fibrosis.

Stem Cell–Conditioned Medium Promotes Graft Remodeling of Midsubstance and Intratunnel Incorporation After Anterior Cruciate Ligament Reconstruction in a Rat Model

Background: Stem cell–conditioned medium (CM) has been increasingly used in regenerative medicine. However, its effect on graft-host integration after anterior cruciate ligament (ACL) reconstruction (ACLR) remains unclear. Purpose: To examine the effect of human bone marrow stem cell (hBMSC)–CM on graft-bone integration and graft midsubstance ligamentization in a rat model of ACLR. Study Design: Controlled laboratory study. Methods: CM was obtained from the supernatant of commercially available hBMSCs in serum-free Dulbecco’s modified Eagle medium (DMEM). In a rat model of an ACL injury, isometric ACLR was performed. Three groups were established: CM injection group (CM; n = 40), control injection group (CI; n = 40) with serum-free DMEM injections, and no injection group (NI; n = 40). An intra-articular injection was performed weekly. Micro–computed tomography was conducted at 2, 4, and 8 weeks postoperatively. Histological and biomechanical analyses were conducted at 4 and 8 weeks postoperatively. The NIH3T3 fibroblast was utilized as a model in vitro to examine the effect of CM using the cell counting kit–8 (CCK-8) assay and immunofluorescence staining of Ki-67, α–smooth muscle actin (α-SMA), and collagen 1 (Col 1). Results: At 4 and 8 weeks, the femoral and tibial bone tunnel areas as well as the interface between the graft and host bone were smaller, while the bone volume/total volume ratio was higher, in the CM group. Sharpey-like fibers formed at 8 weeks in the CM group. At 4 and 8 weeks, more Col 1 was noticed in the CM group than in the NI group (both P < .001) or CI group (both P < .001). Immunohistochemically, the α-SMA–positive area was up-regulated at the graft-bone interface at 4 weeks (P < .001) and declined at 8 weeks (P < .001) in the CM group compared with the other 2 groups. At the midsubstance, α-SMA expression decreased from 4 to 8 weeks in all groups and was significantly lower in the CM group than in the NI group (P < .01) or CI group (P < .05) at 8 weeks. The CCK-8 assay showed that CM increased NIH3T3 viability (P < .001) and the level of Ki-67 (P < .05), α-SMA (P < .001), and Col 1 (P < .001) in CM-educated NIH3T3 cells. Conclusion: hBMSC-CM accelerates graft-bone incorporation and midsubstance ligamentization and enhances the proliferation, differentiation, and collagen synthesis of fibroblasts. Clinical Relevance: Graft-host integration is essential after ACLR. The current study identified a novel agent, that is, hBMSC-CM, as a candidate for promoting integration.

Frontline Science: Reprogramming COX-2, 5-LOX, and CYP4A-mediated arachidonic acid metabolism in macrophages by salidroside alleviates gouty arthritis.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and cytochrome P450 (CYP) 4A-mediated arachidonic acid (AA) metabolism play an essential role in human inflammatory disorders. Blocking COX-2 pathway would shunt AA metabolism to the other pathway, thereby decreasing the efficacy and exacerbating adverse effects. Here we demonstrated that reprogramming COX-2, 5-LOX, and CYP4A-mediated AA metabolism in macrophages by salidroside (Sal) ameliorates monosodium urate (MSU) crystal-induced inflammation. Compared with COX-2 inhibitor celecoxib, Sal (80mg/kg) presented a superior anti-arthritic profile in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, 5-LOX, and CYP4A and production of prostaglandin E2 (PGE2 ), leukotriene B4 (LTB4 ), and 20-hydroxyeicosatetraenoic acid (20-HETE) in the synovial fluid macrophages. Sal decreased representative M1 marker (iNOS and CD86, etc.) expression and M1 cytokine (TNF-α and IL-1β) production, whereas it increased M2 marker (CD206 and Arg-1) expression and M2 cytokine (TGF-β and IL-10) production. The injection of conditioned medium from MSU crystal-treated macrophages into the ankle joint of rats reproduced the gouty inflammation, which was attenuated by Sal. Mechanistically, down-regulation of COX-2, 5-LOX, and CYP4A in the RAW264.7 and NR8383 macrophages by Sal skewed macrophage polarization away from the M1 phenotype, and thereby prevented neutrophil migration and chondrocyte degradation with STAT1 and NF-κB inactivation. Conversely, overexpression of COX-2, 5-LOX, CYP4A or STAT1, or exogenous addition of IL-1β or TNF-α partially abolished these effects. Together, inhibition of COX-2, 5-LOX, and CYP4A in macrophages by Sal ameliorates MSU crystal-induced inflammation through decreasing TNF-α and IL-1β production, and may serve as a novel therapeutic strategy.

Hypoxic conditioned medium derived from bone marrow mesenchymal stromal cells protects against ischemic stroke in rats.

In recent years, studies have shown that the secretome of bone marrow mesenchymal stromal cells (BMSCs) contains many growth factors, cytokines, and antioxidants, which may provide novel approaches to treat ischemic diseases. Furthermore, the secretome may be modulated by hypoxic preconditioning. We hypothesized that conditioned medium (CM) derived from BMSCs plays a crucial role in reducing tissue damage and improving neurological recovery after ischemic stroke and that hypoxic preconditioning of BMSCs robustly improves these activities. Rats were subjected to ischemic stroke by middle cerebral artery occlusionand then intravenously administered hypoxic CM, normoxic CM, or Dulbecco modified Eagle medium (DMEM, control). Cytokine antibody arrays and label-free quantitative proteomics analysis were used to compare the differences between hypoxic CM and normoxic CM. Injection of normoxic CM significantly reduced the infarct area and improved neurological recovery after stroke compared with administering DMEM. These outcomes may be associated with the attenuation of apoptosis and promotion of angiogenesis. Hypoxic preconditioning significantly enhanced these therapeutic effects. Fourteen proteins were significantly increased in hypoxic CM compared with normoxic CM as measured by cytokine arrays. The label-free quantitative proteomics analysis revealed 163 proteins that were differentially expressed between the two groups, including 107 upregulated proteins and 56 downregulated proteins. Collectively, our results demonstrate that hypoxic CM protected brain tissue from ischemic injury and promoted functional recovery after stroke in rats and that hypoxic CM may be the basis of a potential therapy for stroke patients.

Effects of Intrathecal Injection of the Conditioned Medium from Bone Marrow Stromal Cells on Spinal Cord Injury in Rats.

Bone marrow stromal cells (BMSCs) have been studied for the treatment of spinal cord injury (SCI). In previous studies, we showed that the transplantation of BMSCs, even though they disappeared from the host spinal cord within 1-3 weeks after transplantation, improved locomotor behaviors and promoted axonal regeneration. This result led to the hypothesis that BMSCs might release some neurotrophic factors effective for the treatment of SCI. The present study examined this by injecting the conditioned medium (CM) of BMSCs to treat SCI in rats. The spinal cord was contusion-injured, followed immediately by continuous injection for 2 weeks of the CM of BMSCs through the cerebrospinal fluid via the 4th ventricle using an Alzet osmotic pump. Locomotor behaviors evaluated by the Basso-Beattie-Bresnahan score were markedly improved in the CM-injection group, compared with the control group, at 1 to 4 weeks post-injection. The contusion-injured site of the spinal cord was identified as an astrocyte-devoid area, which contained no astrocytes but was filled with collagen matrices and empty cavities of various sizes. Collagen matrices contained type I collagen and laminin. Numerous axons extended through the collagen matrices of the astrocyte-devoid area. Axons were surrounded by Schwann cells, exhibiting the same morphological characteristics as peripheral nerve fibers. The density of axons extending through the astrocyte-devoid area was higher in the CM-injection group, compared with the control group. CM injection had beneficial effects on locomotor improvements and tissue repair, including axonal regeneration, meaning that the BMSC-CM stimulated the intrinsic ability of the spinal cord to regenerate. Activation of the intrinsic ability of the spinal cord to regenerate by the injection of neurotrophic factors such as BMSC-CM is considered to be a safe and preferable method for the clinical treatment of SCI.

Aspirin blocks cancer-associated cognitive impairment

Over 50% of breast cancer patients develop chronic cognitive impairment including concentration, learning and memory difficulties. This has been attributed to the effects of chemotherapy, but recent evidence indicates that cognitive impairment is present in cancer patients prior to any treatment, suggesting the tumor itself may be responsible. To develop novel treatments to block cancer-associated cognitive impairment, it is imperative to identify the mechanisms responsible. To determine if breast cancer is sufficient to induce cognitive impairment, we injected luciferase-tagged syngenic mammary adenocarcinoma cells (1 × 105 4T1.2 cells) vs. vehicle into BALB/c female mice, and assessed episodic memory and sickness. Tumor-bearing mice had significantly poorer episodic memory than non-tumor- bearing mice from 3 days as measured by novel object and social recognition. Sickness (body weight loss, reduced burrowing and locomotor activity) only surfaced during late metastatic burden (from 24 days). Proinflammatory cytokines were elevated in tumor-bearing mice and injection of conditioned media from tumor cells was sufficient to induce memory impairment, suggesting that soluble factors secreted from tumors are responsible for these effects. Significantly, treatment with low-dose anti-inflammatory drugs (aspirin vs placebo in drinking water, equivalent to 100 mg/day in humans) completely blocked tumor- induced episodic memory impairment without affecting tumor-induced sickness or tumor growth. These data suggest that safe, available anti-inflammatories may be repurposed to prevent and treat cognitive impairment in cancer patients and survivors.

A Novel Role for CAMKK1 in the Regulation of the Mesenchymal Stem Cell Secretome.

Transplantation of adult stem cells into myocardial tissue after acute myocardial infarction (AMI), has been shown to improve tissue recovery and prevent progression to ischemic cardiomyopathy. Studies suggest that the effects of mesenchymal stem cells (MSC) are due to paracrine factors released by MSC, as the benefits of MSC can be achieved through delivery of conditioned media (CM) alone. We previously demonstrated that downregulation of Dab2 enhances MSC cardiac protein expression and improves cardiac function after AMI following MSC engraftment. In order to define the molecular mechanisms that regulate MSC secretome, we analyzed gene arrays in MSC following downregulation of Dab2 via TGFβ1 pretreatment or transfection with Dab2:siRNA or miR‐145. We identified 23 genes whose expressions were significantly changed in all three conditions. Among these genes, we have initially focused our validation and functional work on calcium/calmodulin‐dependent protein kinase kinase‐1 (CAMKK1). We quantified the effects of CAMKK1 overexpression in MSC following injection of CM after AMI. Injections of CM from MSC with CAMKK1 over‐expression correlated with an increase in vascular density (CAMKK1 CM: 2,794.95 ± 44.2 versus Control: 1,290.69 ± 2.8 vessels/mm2) and decreased scar formation (CAMKK1 CM 50% ± 3.2% versus Control: 28% ± 1.4%), as well as improved cardiac function. Direct overexpression of CAMKK1 in infarcted tissue using a CAMKK1‐encoding plasmid significantly improved ejection fraction (CAMKK1: 83.2% ± 5.4% versus saline: 51.7% ± 5.8%. Baseline: 91.3% ± 4.3%) and decreased infarct size after AMI. Our data identify a novel role for CAMKK1 as regulator of the MSC secretome and demonstrate that direct overexpression of CAMKK1 in infarcted cardiac tissue, results in therapeutic beneficial effects. Stem Cells Translational Medicine 2017;6:1759–1766

Treatment with adipose derived mesenchymal stem cells and their conditioned media reverse carrageenan induced paw oedema in db/db mice

Mesenchymal stem cells are known for anti inflammatory and immunomodulatory activities. The aim of our study was to evaluate the effect of human adipose derived mesenchymal stem cells (hADMSCs) and its conditioned media (CM) on carrageenan induced acute inflammation in db/db mice. We injected 5×105 ADMSCs or the CM in the inflamed paw. We assessed the paw volume, serum IL6 levels and histopathology of the paw to reveal the anti inflammatory effect. We observed a single injection of hADMSCs or CM could reverse the inflammation within 24h as evidenced by reduction in paw volume, IL6 levels and histological examination. Our result equivocally demonstrates the role of CM in normalising the inflammation better than hADMSCs. This study will pave way for an alternative to anti inflammatory drugs.

Surfactant Protein B Suppresses Lung Cancer Progression by Inhibiting Secretory Phospholipase A2 Activity and Arachidonic Acid Production

Background/Aims: Radiotherapy is applied to patients with inoperable cancer types including advanced stage non-small cell lung cancer (NSCLC) and radioresistance functions as a critical obstacle in radiotherapy. This study was aimed to investigate the mechanism of radioresistance regulated by surfactant protein B (SP-B). Methods: To investigate the role of SP-B in radioresistance, ΔSFTPB A549 cell line was established and SP-B expression was analyzed. In response to ionizing radiation (IR), the change of SP-B expression was analyzed in A549 and NCI-H441 cell lines. Conditioned media (CM) from NSCLC cells were utilized to evaluate the downstream signaling pathway. The in vivo effects of SP-B were assessed through mouse xenograft model with intratumoral injection of CM. Results: In response to IR, NSCLC cell lines showed decreased SP-B regulated by the TGF-β signaling and decreased SP-B stimulated cell survival and epithelial-mesenchymal transition. Treatment with CM from irradiated cells activated sPLA₂, enhanced protein kinase Cδ-MAPKs signaling pathway, and increased arachidonic acid production. We confirmed the in vivo roles of SP-B through mouse xenograft model. Conclusion: Our results revealed that down-regulation of SP-B was involved in the radiation-induced metastatic conversion of NSCLC and provided evidence that SP-B acted as a suppressor of NSCLC progression.

Secretions from placenta, after hypoxia/reoxygenation, can damage developing neurones of brain under experimental conditions

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca2+]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.

Delayed Administration of Bone Marrow Mesenchymal Stem Cell Conditioned Medium Significantly Improves Outcome After Retinal Ischemia in Rats

Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-μm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect.

Conditioned medium from amniotic membrane-derived cells prevents lung fibrosis and preserves blood gas exchanges in bleomycin-injured mice—specificity of the effects and insights into possible mechanisms

Background and aimsWe recently demonstrated that injection of conditioned medium (CM) generated from cells of the mesenchymal region of human amniotic membrane (AMTCs) reduces bleomycin-induced lung fibrosis in mice, suggesting a crucial role of paracrine factor(s) secreted by AMTCs in these beneficial effects. We further investigated this hypothesis, the mechanisms involved, the effects on some lung functional parameters and whether AMTC-secreted effector(s) are specific to these cells and not produced by other cell types, extending the time of analysis up to 28 days after treatment. MethodsBleomycin-challenged mice were either treated with AMTC-CM or CM generated from human skin fibroblasts, human peripheral blood mononuclear cells or Jurkat cells, or were left untreated. Mouse lungs were analyzed for content of pro-inflammatory and pro-fibrotic molecules, presence of lymphocytes and macrophages and for fibrosis level (through histological semi-quantitative evaluation and quantitative measurement of collagen content). Arterial blood gas analysis was also performed. ResultsUp to 28 days after delivery, AMTC-CM-treated mice developed reduced lung fibrosis with respect to mice treated with other CM types. AMTC-CM-treated mice had comparatively better preservation of blood gas parameters and showed lower lung content of interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1 and transforming growth factor-β associated with reduced lung macrophage levels. ConclusionsAMTC-CM prevents lung fibrosis in bleomycin-challenged mice, improving survival and preserving lung functional parameters such as blood gas exchanges. The specificity of AMTC-CM action was indicated by the absence of fibrosis reduction when other CM types were used. Finally, we provide some insights into the possible mechanisms underlying AMTC-CM-mediated control of fibrosis.