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

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

Acute aortic dissection (AAD) is a serious disease in which the largest artery in the body, called the aorta, enlarges and ruptures. Surgery is often required to prevent death. Cells called Muse cells have been injected into people during clinical trials to treat other diseases. In this study, we injected Muse cells into mice with dissected aorta. The cells accumulated in damaged parts of the aorta and strengthened the structure of the aorta, reducing the number of mice that died. If further research shows this treatment works in humans, this could enable AAD to be treated without surgery and potentially improve the treatment and survival of people with AAD.

New rat model of spinal cord infarction with long-lasting functional disabilities generated by intraspinal injection of endothelin-1.

BACKGROUND: The current method for generating an animal model of spinal cord (SC) infarction is highly invasive and permits only short-term observation, typically limited to 28 days. OBJECTIVE: We aimed to establish a rat model characterised by long-term survival and enduring SC dysfunction by inducing selective ischaemic SC damage. METHODS: In 8-week-old male Wistar rats, a convection-enhanced delivery technique was applied to selectively deliver endothelin-1 (ET-1) to the anterior horn of the SC at the Th13 level, leading to SC infarction. The Basso, Beattie and Bresnahan (BBB) locomotor score was assessed for 56 days. The SC was examined by a laser tissue blood flowmeter, MRI, immunohistochemistry, triphenyl tetrazolium chloride (TTC) staining, Western blots and TUNEL staining. RESULTS: The puncture method was used to bilaterally inject 0.7 µL ET-1 (2.5 mg/mL) from the lateral SC into the anterior horns (40° angle, 1.5 mm depth) near the posterior root origin. Animals survived until day 56 and the BBB score was stably maintained (5.5±1.0 at day 14 and 6.2±1.0 at day 56). Rats with BBB scores ≤1 on day 1 showed stable scores of 5-6 after day 14 until day 56 while rats with BBB scores >1 on day 1 exhibited only minor dysfunction with BBB scores >12 after day 14. TTC staining, immunostaining and TUNEL staining revealed selective ischaemia and neuronal cell death in the anterior horn. T2-weighted MR images showed increasing signal intensity at the SC infarction site over time. Western blots revealed apoptosis and subsequent inflammation in SC tissue after ET-1 administration. CONCLUSIONS: Selective delivery of ET-1 into the SC allows for more precise localisation of the infarcted area at the targeted site and generates a rat SC infarction model with stable neurological dysfunction lasting 56 days.

Tumor suppressor let-7 acts as a key regulator for pluripotency gene expression in Muse cells.

In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the expression of an RNA-binding pluripotency-relevant protein, LIN28, and the absence of its antagonist, the tumor-suppressor microRNA (miRNA) let-7, play a key role in maintaining pluripotency. Muse cells are non-tumorigenic pluripotent-like stem cells residing in the bone marrow, peripheral blood, and organ connective tissues as pluripotent surface marker SSEA-3(+). They express pluripotency genes, differentiate into triploblastic-lineage cells, and self-renew at the single cell level. Muse cells do not express LIN28 but do express let-7 at higher levels than in iPSCs. In Muse cells, we demonstrated that let-7 inhibited the PI3K-AKT pathway, leading to sustainable expression of the key pluripotency regulator KLF4 as well as its downstream genes, POU5F1, SOX2, and NANOG. Let-7 also suppressed proliferation and glycolysis by inhibiting the PI3K-AKT pathway, suggesting its involvement in non-tumorigenicity. Furthermore, the MEK/ERK pathway is not controlled by let-7 and may have a pivotal role in maintaining self-renewal and suppression of senescence. The system found in Muse cells, in which the tumor suppressor let-7, but not LIN28, tunes the expression of pluripotency genes, might be a rational cell system conferring both pluripotency-like properties and a low risk for tumorigenicity.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations.

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Phagocytosing differentiated cell-fragments is a novel mechanism for controlling somatic stem cell differentiation within a short time frame.

Stem cells undergo cytokine-driven differentiation, but this process often takes longer than several weeks to complete. A novel mechanism for somatic stem cell differentiation via phagocytosing 'model cells' (apoptotic differentiated cells) was found to require only a short time frame. Pluripotent-like Muse cells, multipotent mesenchymal stem cells (MSCs), and neural stem cells (NSCs) phagocytosed apoptotic differentiated cells via different phagocytic receptor subsets than macrophages. The phagocytosed-differentiated cell-derived contents (e.g., transcription factors) were quickly released into the cytoplasm, translocated into the nucleus, and bound to promoter regions of the stem cell genomes. Within 24 ~ 36 h, the cells expressed lineage-specific markers corresponding to the phagocytosed-differentiated cells, both in vitro and in vivo. At 1 week, the gene expression profiles were similar to those of the authentic differentiated cells and expressed functional markers. Differentiation was limited to the inherent potential of each cell line: triploblastic-, adipogenic-/chondrogenic-, and neural-lineages in Muse cells, MSCs, and NSCs, respectively. Disruption of phagocytosis, either by phagocytic receptor inhibition via small interfering RNA or annexin V treatment, impeded differentiation in vitro and in vivo. Together, our findings uncovered a simple mechanism by which differentiation-directing factors are directly transferred to somatic stem cells by phagocytosing apoptotic differentiated cells to trigger their rapid differentiation into the target cell lineage.

Naïve pluripotent-like characteristics of non-tumorigenic Muse cells isolated from human amniotic membrane.

Multilineage-differentiating stress-enduring (Muse) cells are non-tumorigenic pluripotent-like stem cells that exhibit triploblastic differentiation and self-renewability at the single-cell level, and are collectable as pluripotent surface marker SSEA-3(+) from the bone marrow (BM), peripheral blood, and organ connective tissues. SSEA-3(+) cells from human amniotic membrane mesenchymal stem cells (hAMSCs) were compared with hBM-Muse cells. Similar to hBM-Muse cells, hAMSC-SSEA-3(+) cells expressed pluripotency genes (OCT3/4, NANOG, and SOX2), differentiated into triploblastic cells from a single cell, self-renewed, and exhibited non-tumorigenicity. Notably, however, they exhibited unique characteristics not seen in hBM-Muse cells, including higher expression of genes related to germline- and extraembryonic cell-lineages compared with those in hBM-Muse cells in single-cell RNA-sequencing; and enhanced expression of markers relevant to germline- (PRDM14, TFAP2C, and NANOS3) and extraembryonic cell- (CDX2, GCM1, and ID2) lineages when induced by cytokine subsets, suggesting a broader differentiation potential similar to naïve pluripotent stem cells. t-SNE dimensionality reduction and Gene ontology analysis visualized hAMSC-SSEA-3(+) cells comprised a large undifferentiated subpopulation between epithelial- and mesenchymal-cell states and a small mesenchymal subpopulation expressing genes relevant to the placental formation. The AM is easily accessible by noninvasive approaches. These unique cells are a potentially interesting target naïve pluripotent stem cell-like resource without tumorigenicity.

Inhibition of Gap Junctional Intercellular Communication Upregulates Pluripotency Gene Expression in Endogenous Pluripotent Muse Cells.

Gap junctions (GJ) are suggested to support stem cell differentiation. The Muse cells that are applied in clinical trials are non-tumorigenic pluripotent-like endogenous stem cells, can be collected as stage-specific embryonic antigen 3 (SSEA-3+) positive cells from multiple tissues, and show triploblastic differentiation and self-renewability at a single cell level. They were reported to up-regulate pluripotency gene expression in suspension. We examined how GJ inhibition affected pluripotency gene expression in adherent cultured-Muse cells. Muse cells, mainly expressing gap junction alpha-1 protein (GJA1), reduced GJ intercellular communication from ~85% to 5-8% after 24 h incubation with 120 µM 18α-glycyrrhetinic acid, 400 nM 12-O-tetradecanoylphorbol-13-acetate, and 90 µM dichlorodiphenyltrichloroethane, as confirmed by a dye-transfer assay. Following inhibition, NANOG, OCT3/4, and SOX2 were up-regulated 2-4.5 times more; other pluripotency-related genes, such as KLF4, CBX7, and SPRY2 were elevated; lineage-specific differentiation-related genes were down-regulated in quantitative-PCR and RNA-sequencing. Connexin43-siRNA introduction also confirmed the up-regulation of NANOG, OCT3/4, and SOX2. YAP, a co-transcriptional factor in the Hippo signaling pathway that regulates pluripotency gene expression, co-localized with GJA1 (also known as Cx43) in the cell membrane and was translocated to the nucleus after GJ inhibition. Adherent culture is usually more suitable for the stable expansion of cells than is a suspension culture. GJ inhibition is suggested to be a simple method to up-regulate pluripotency in an adherent culture that involves a Cx43-YAP axis in pluripotent stem cells, such as Muse cells.

Human Muse cells reduce myocardial infarct size and improve cardiac function without causing arrythmias in a swine model of acute myocardial infarction.

BACKGROUND: We recently reported that multilineage-differentiating stress enduring (Muse) cells intravenously administered after acute myocardial infarction (AMI), selectively engrafted to the infarct area, spontaneously differentiated into cardiomyocytes and vessels, reduced the infarct size, improved the left ventricular (LV) function and remodeling in rabbits. We aimed to clarify the efficiency of Muse cells in a larger animal AMI model of mini-pigs using a semi-clinical grade human Muse cell product. METHOD AND RESULT: Mini-pigs underwent 30 min of coronary artery occlusion followed by 2 weeks of reperfusion. Semi-clinical grade human Muse cell product (1x107, Muse group, n = 5) or saline (Vehicle group, n = 7) were intravenously administered at 24 h after reperfusion. The infarct size, LV function and remodeling were evaluated by echocardiography. Arrhythmias were evaluated by an implantable loop recorder. The infarct size was significantly smaller in the Muse group (10.5±3.3%) than in the Vehicle group (21.0±2.0%). Both the LV ejection fraction and fractional shortening were significantly greater in the Muse group than in the Vehicle group. The LV end-systolic and end-diastolic dimensions were significantly smaller in the Muse group than in the Vehicle group. Human Muse cells homed into the infarct border area and expressed cardiac troponin I and vascular endothelial CD31. No arrhythmias and no blood test abnormality were observed. CONCLUSION: Muse cell product might be promising for AMI therapy based on the efficiency and safety in a mini-pig AMI.

Intravenous injection of human multilineage-differentiating stress-enduring cells alleviates mouse severe acute pancreatitis without immunosuppressants.

INTRODUCTION: We examined the effect of intravenously injected human multilineage-differentiating stress-enduring (Muse) cells, non-tumorigenic endogenous reparative stem cells already used in clinical trials, on a severe acute pancreatitis (SAP) mouse model without immunosuppressants. METHODS: Human Muse cells (1.0 × 105 cells) collected from mesenchymal stem cells (MSCs) as SSEA-3(+) were injected into a C57BL/6 mouse model via the jugular vein 6 h after SAP-induction with taurocholate. The control group received saline or the same number of SSEA-3(-)-non-Muse MSCs. RESULTS: Edematous parameters, F4/80(+) macrophage infiltration and terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity was the lowest and the number of proliferating endogenous pancreatic progenitors (CK18(+)/Ki67(+) cells) the highest in the Muse group among the three groups, with statistical significance, at 72 h. An enzyme-linked immunosorbent assay and quantitative polymerase chain reaction demonstrated that in vitro production of VEGF, HGF, IGF-1, and MMP-2, which are relevant to tissue protection, anti-inflammation, and anti-fibrosis, were higher in Muse cells than in non-Muse MSCs, particularly when cells were cultured in SAP mouse serum. Consistently, the pancreas of animals in the Muse group contained higher amounts of those factors according to Western blotting at 18 h than that in the non-Muse MSCs and control groups. CONCLUSIONS: Intravenous injection of human Muse cells was suggested to be effective for attenuating edema, inflammation and apoptosis in the acute phase of SAP.

Isolation and characterization of bone marrow-derived mesenchymal stem cells in Xenopus laevis.

Mesenchymal stem cells (MSCs) are multipotent cells that exist in mesenchymal tissues such as bone marrow and are able to differentiate into osteocytes, chondrocytes, and adipocytes. MSCs are generally collected as adherent cells on a plastic dish, and are positive for markers such as CD44, CD73, CD90, CD105 and CD166, and negative for CD11b, CD14, CD19, CD31, CD34, CD45, CD79a and HLA-DR. MSCs have been established from many kinds of mammals, but MSCs from amphibians have not yet been reported. We cultured adherent cells from the bone marrow of Xenopus laevis by modifying the protocol for culturing mammalian MSCs. The morphology of these cells was similar to that of mammalian MSCs. The amphibian MSCs were positive for cd44, cd73, cd90 and cd166, and negative for cd11b, cd14, cd19, cd31, cd34, cd45, cd79a and hla-dra. Moreover, they could be induced to differentiate into osteocyte-, chondrocyte-, and adipocyte-lineage cells by cytokine induction systems that were similar to those used for mammalian MSC differentiation. Thus, they are considered to be similar to mammalian MSCs. Unlike mammals, amphibians have high regenerative capacity. The findings from the present study will allow for future research to reveal how Xenopus MSCs are involved in the amphibian regenerative capacity and to elucidate the differences in the regenerative capacity between mammals and amphibians.

Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle.

Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs.

Protection of liver sinusoids by intravenous administration of human Muse cells in a rat extra-small partial liver transplantation model.

Small-for-size syndrome (SFSS) has a poor prognosis due to excessive shear stress and sinusoidal microcirculatory disturbances in the acute phase after living-donor liver transplantation (LDLT). Multilineage-differentiating stress enduring (Muse) cells are reparative stem cells found in various tissues and currently under clinical trials. These cells selectively home to damaged sites via the sphingosine-1-phosphate (S1P)-S1P receptor 2 system and repair damaged tissue by pleiotropic effects, including tissue protection and damaged/apoptotic cell replacement by differentiating into tissue-constituent cells. The effects of intravenously administered human bone marrow-Muse cells and -mesenchymal stem cells (MSCs) (4 × 105 ) on liver sinusoidal endothelial cells (LSECs) were examined in a rat SFSS model without immunosuppression. Compared with MSCs, Muse cells intensively homed to the grafted liver, distributed to the sinusoids and vessels, and delivered improved blood chemistry and Ki-67(+) proliferative hepatocytes and -LSECs within 3 days. Tissue clearing and three-dimensional imaging by multiphoton laser confocal microscopy revealed maintenance of the sinusoid continuity, organization, and surface area, as well as decreased sinusoid interruption in the Muse group. Small-interfering RNA-induced knockdown of hepatocyte growth factor and vascular endothelial growth factor-A impaired the protective effect of Muse cells on LSECs. Intravenous injection of Muse cells might be a feasible approach for LDLT with less recipient burden.

Intravenously delivered multilineage-differentiating stress enduring cells dampen excessive glutamate metabolism and microglial activation in experimental perinatal hypoxic ischemic encephalopathy.

Perinatal hypoxic ischemic encephalopathy (HIE) results in serious neurological dysfunction and mortality. Clinical trials of multilineage-differentiating stress enduring cells (Muse cells) have commenced in stroke using intravenous delivery of donor-derived Muse cells. Here, we investigated the therapeutic effects of human Muse cells in an HIE model. Seven-day-old rats underwent ligation of the left carotid artery then were exposed to 8% oxygen for 60 min, and 72 hours later intravenously transplanted with 1 × 104 of human-Muse and -non-Muse cells, collected from bone marrow-mesenchymal stem cells as stage-specific embryonic antigen-3 (SSEA-3)+ and -, respectively, or saline (vehicle) without immunosuppression. Human-specific probe revealed Muse cells distributed mainly to the injured brain at 2 and 4 weeks, and expressed neuronal and glial markers until 6 months. In contrast, non-Muse cells lodged in the lung at 2 weeks, but undetectable by 4 weeks. Magnetic resonance spectroscopy and positron emission tomography demonstrated that Muse cells dampened excitotoxic brain glutamatergic metabolites and suppressed microglial activation. Muse cell-treated group exhibited significant improvements in motor and cognitive functions at 4 weeks and 5 months. Intravenously transplanted Muse cells afforded functional benefits in experimental HIE possibly via regulation of glutamate metabolism and reduction of microglial activation.

The evaluation of the safety and efficacy of intravenously administered allogeneic multilineage-differentiating stress-enduring cells in a swine hepatectomy model.

INTRODUCTION: Multilineage-differentiating stress-enduring (Muse) cells are non-tumorigenic endogenous pluripotent-like cells residing in the bone marrow that exert a tissue reparative effect by replacing damaged/apoptotic cells through spontaneous differentiation into tissue-constituent cells. Post-hepatectomy liver failure (PHLF) is a potentially fatal complication. The main purpose of this study was to evaluate the safety and efficiency of allogeneic Muse cell administration via the portal vein in a swine model of PHLF. METHODS: Swine Muse cells, collected from swine bone marrow-mesenchymal stem cells (MSCs) as SSEA-3(+) cells, were examined for their characteristics. Then, 1 × 107 allogeneic-Muse cells and allogeneic-MSCs and vehicle were injected via the portal vein in a 70% hepatectomy swine model. RESULTS: Swine Muse cells exhibited characteristics comparable to previously reported human Muse cells. Compared to the MSC and vehicle groups, the Muse group showed specific homing of the administered cells into the liver, resulting in improvements in the control of hyperbilirubinemia (P = 0.04), prothrombin international normalized ratio (P = 0.05), and suppression of focal necrosis (P = 0.04). Integrated Muse cells differentiated spontaneously into hepatocyte marker-positive cells. CONCLUSIONS: Allogeneic Muse cell administration may provide a reparative effect and functional recovery in a 70% hepatectomy swine model and thus may contribute to the treatment of PHLF.

Therapeutic benefit of Muse cells in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron loss. Muse cells are endogenous reparative pluripotent-like stem cells distributed in various tissues. They can selectively home to damaged sites after intravenous injection by sensing sphingosine-1-phosphate produced by damaged cells, then exert pleiotropic effects, including tissue protection and spontaneous differentiation into tissue-constituent cells. In G93A-transgenic ALS mice, intravenous injection of 5.0 × 104 cells revealed successful homing of human-Muse cells to the lumbar spinal cords, mainly at the pia-mater and underneath white matter, and exhibited glia-like morphology and GFAP expression. In contrast, such homing or differentiation were not recognized in human mesenchymal stem cells but were instead distributed mainly in the lung. Relative to the vehicle groups, the Muse group significantly improved scores in the rotarod, hanging-wire and muscle strength of lower limbs, recovered the number of motor neurons, and alleviated denervation and myofiber atrophy in lower limb muscles. These results suggest that Muse cells homed in a lesion site-dependent manner and protected the spinal cord against motor neuron death. Muse cells might also be a promising cell source for the treatment of ALS patients.

A Novel Type of Stem Cells Double-Positive for SSEA-3 and CD45 in Human Peripheral Blood.

Peripheral blood (PB) contains several types of stem/progenitor cells, including hematopoietic stem and endothelial progenitor cells. We identified a population positive for both the pluripotent surface marker SSEA-3 and leukocyte common antigen CD45 that comprises 0.04% ± 0.003% of the mononuclear cells in human PB. The average size of the SSEA-3(+)/CD45(+) cells was 10.1 ± 0.3 µm and ∼22% were positive for CD105, a mesenchymal marker; ∼85% were positive for CD19, a B cell marker; and ∼94% were positive for HLA-DR, a major histocompatibility complex class II molecule relevant to antigen presentation. These SSEA-3(+)/CD45(+) cells expressed the pluripotency markers Nanog, Oct3/4, and Sox2, as well as sphingosine-1-phosphate (S1P) receptor 2, and migrated toward S1P, although their adherence and proliferative activities in vitro were low. They expressed NeuN at 7 d, Pax7 and desmin at 7 d, and alpha-fetoprotein and cytokeratin-19 at 3 d when supplied to mouse damaged tissues of the brain, skeletal muscle and liver, respectively, suggesting the ability to spontaneously differentiate into triploblastic lineages compatible to the tissue microenvironment. Multilineage-differentiating stress enduring (Muse) cells, identified as SSEA-3(+) in tissues such as the bone marrow and organ connective tissues, express pluripotency markers, migrate to sites of damage via the S1P-S1P receptor 2 system, and differentiate spontaneously into tissue-compatible cells after homing to the damaged tissue where they participate in tissue repair. After the onset of acute myocardial infarction and stroke, patients are reported to have an increase in the number of SSEA-3(+) cells in the PB. The SSEA-3(+)/CD45(+) cells in the PB showed similarity to tissue-Muse cells, although with difference in surface marker expression and cellular properties. Thus, these findings suggest that human PB contains a subset of cells that are distinct from known stem/progenitor cells, and that CD45(+)-mononuclear cells in the PB comprise a novel subpopulation of cells that express pluripotency markers.

Intravenously Transplanted Human Multilineage-Differentiating Stress-Enduring Cells Afford Brain Repair in a Mouse Lacunar Stroke Model.

Background and Purpose- Multilineage-differentiating stress-enduring cells are endogenous nontumorigenic reparative pluripotent-like stem cells found in bone marrow, peripheral blood, and connective tissues. Topically administered human multilineage-differentiating stress-enduring cells into rat/mouse stroke models differentiated into neural cells and promoted clinically relevant functional recovery. However, critical questions on the appropriate timing and dose, and safety of the less invasive intravenous administration of clinical-grade multilineage-differentiating stress-enduring cell-based product CL2020 remain unanswered. Methods- Using an immunodeficient mouse lacunar model, CL2020 was administered via the cervical vein in different doses (high dose=5×104 cells/body; medium dose=1×104 cells/body; low dose=5×103 cells/body) at subacute phase (≈9 days after onset) and chronic phase (≈30 days). Cylinder test, depletion of human cells by diphtheria toxin administration, immunohistochemistry, and human specific-genome detection were performed. Results- Tumorigenesis and adverse effects were not detected for up to 22 weeks. The high-dose group displayed significant functional recovery compared with the vehicle group in cylinder test in subacute-phase-treated and chronic-phase-treated animals after 6 weeks and 8 weeks post-injection, respectively. In the high-dose group of subacute-phase-treated animals, robust and stable recovery in cylinder test persisted up to 22 weeks compared with the vehicle group. In both groups, intraperitoneal injection of diphtheria toxin abrogated the functional recovery. Anti-human mitochondria revealed CL2020 distributed mainly in the peri-infarct area at 1, 10, and 22 weeks and expressed NeuN (neuronal nuclei)- and MAP-2 (microtubule-associated protein-2)-immunoreactivity. Conclusions- Intravenously administered CL2020 was safe, migrated to the peri-infarct area, and afforded functional recovery in experimental stroke.

Rescue from Stx2-Producing E. coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Direct conversion of adult human skin fibroblasts into functional Schwann cells that achieve robust recovery of the severed peripheral nerve in rats.

Direct conversion is considered a promising approach to obtain tissue-specific cells for cell therapies; however, this strategy depends on exogenous gene expression that may cause undesired adverse effects such as tumorigenesis. By optimizing the Schwann cell induction system, which was originally developed for trans-differentiation of bone marrow mesenchymal stem cells into Schwann cells, we established a system to directly convert adult human skin fibroblasts into cells comparable to authentic human Schwann cells without gene introduction. Serial treatments with beta-mercaptoethanol, retinoic acid, and finally a cocktail of basic fibroblast growth factor, forskolin, platelet-derived growth factor-AA, and heregulin-ß1 (EGF domain) converted fibroblasts into cells expressing authentic Schwann cell markers at an efficiency of approximately 75%. Genome-wide gene expression analysis suggested the conversion of fibroblasts into the Schwann cell-lineage. Transplantation of induced Schwann cells into severed peripheral nerve of rats facilitated axonal regeneration and robust functional recovery in sciatic function index comparable to those of authentic human Schwann cells. The contributions of induced Schwann cells to myelination of regenerated axons and re-formation of neuromuscular junctions were also demonstrated. Our data clearly demonstrated that cells comparable to functional Schwann cells feasible for the treatment of neural disease can be induced from adult human skin fibroblasts without gene introduction. This direct conversion system will be beneficial for clinical applications to peripheral and central nervous system injuries and demyelinating diseases.