The secretome of MUSE cells contains factors that may play a role in regulation of stemness, apoptosis and immunomodulation

<p>Mesenchymal stromal cells (MSCs) are a heterogeneous population, which contain several cell phenotypes: mesenchymal stem cells, progenitor cells, fibroblasts and other type of cells. Previously, we identified unique stem cells that we named multilineage-differentiating stress enduring (Muse) cells as one to several percent of MSCs of the bone marrow, adipose tissue and dermis. Among different cell populations in MSCs, Muse cells, positive for pluripotent surface marker SSEA-3, may represent cells responsible for pluripotent-like property of MSCs, since they express pluripotency genes, able to differentiated into triploblastic cells from a single cells and are self-renewable.</p> <p>MSCs release biologically active factors that have profound effects on local cellular dynamics. A thorough examination of MSC secretome seems essential for understanding the physiological functions exerted by these cells in our organism and also for rational cellular therapy design. In this setting, studies on secretome of Muse cells may shed light on pathways that are associated with their specific features.</p> <p>Our findings evidenced that secretomes of MSCs and Muse cells contain factors that regulate extracellular matrix remodeling, ox-redox activities and immune system. Muse cells appear to secrete factors that may preserve their stem cell features, allow survival under stress conditions and may contribute to their immunomodulation capacity.</p> <p>In detail, the proteins belonging to protein kinase A signaling, FXR/RXR activation and LXR/RXR activation pathways may play a role in regulation of Muse stem cell features. These last 2 pathways together with proteins associated with antigen presentation pathway and coagulation system may play a role in immunomodulation.</p

RT-PCR and Q-PCR of typical examples in each colonies H∼L and iPS colony G and human ES cells.

<p>(A) RT-PCR analysis examined the expression of endogenous Oct3/4, Sox2, Nanog, Klf4, c-Myc, as well as FoxD3, Rex1, Dnmt3b, Abcg2 and Cdx2. Beta-actin was used as an internal control. (B) Q-PCR data for expression of endogenous Oct3/4, endogenous Sox2, Nanog, endogenous Klf4, endogenous c-Myc, FoxD3, Rex1, Dnmt3b, Abcg2 and Cdx2.</p

Morphologic and Gene Expression Criteria for Identifying Human Induced Pluripotent Stem Cells

<div><p>Induced pluripotent stem (iPS) cells can be generated from somatic cells by the forced expression of four factors, Oct3/4, Sox2, Klf4, and c-Myc. While a great variety of colonies grow during induction, only a few of them develop into iPS cells. Researchers currently use visual observation to identify iPS cells and select colonies resembling embryonic stem (ES) cells, and there are no established objective criteria. Therefore, we exhaustively analyzed the morphology and gene expression of all the colonies generated from human fibroblasts after transfection with four retroviral vectors encoding individual factors (192 and 203 colonies in two experiments) and with a single polycistronic retroviral vector encoding all four factors (199 and 192 colonies in two experiments). Here we demonstrate that the morphologic features of emerged colonies can be categorized based on six parameters, and all generated colonies that could be passaged were classified into seven subtypes in colonies transfected with four retroviral vectors and six subtypes with a single polycistronic retroviral vector, both including iPS cell colonies. The essential qualifications for iPS cells were: cells with a single nucleolus; nucleus to nucleolus (N/Nls) ratio ∼2.19: cell size ∼43.5 µm²: a nucleus to cytoplasm (N/C) ratio ∼0.87: cell density in a colony ∼5900 cells/mm²: and number of cell layer single. Most importantly, gene expression analysis revealed for the first time that endogenous Sox2 and Cdx2 were expressed specifically in iPS cells, whereas Oct3/4 and Nanog, popularly used markers for identifying iPS cells, are expressed in colonies other than iPS cells, suggesting that Sox2 and Cdx2 are reliable markers for identifying iPS cells. Our findings indicate that morphologic parameters and the expression of endogenous Sox2 and Cdx2 can be used to accurately identify iPS cells.</p> </div

Double immunohistochemistry of GFP-Tuj-1 (A and B), GFP-neuronal nuclear antigen (NeuN; C) and human mitochondria-GFAP (D) in ipsilateral cortex of Muse cell-group (42 days after transplantation).

<p>The white square in panel A represents the location of Panel B. Scale bars = 50 μm.</p

Rotarod treadmill performance. Line graph shows the temporal profile of functional recovery in vehicle-, BMSC-, non-Muse cell-, and Muse cell-treated mice subjected to permanent middle cerebral artery occlusion (pMCAO).

<p>**, ††, ¶¶ P<0.01 vs. vehicle-treated mice. Sec indicates seconds.</p

Characterization of iPS cell colony G.

<p>(A–D) Immunocytochemistry for (A) Oct3/4, (B) Nanog, (C) Sox2, (D) TRA-1-81 in iPS cell colony G. Scale bar = 100 µm. (E, F) EBs generated from colony G were plated on gelatin coated dishes containing DMEM/F12 medium supplemented with 20% knockout serum replacement. After 10 days, cell differentiation was confirmed by immunocytochemistry for mesodermal (smooth muscle actin; SMA) (E), endodermal (alpha-fetoprotein; alpha-FP) (E) and ectodermal markers (neurofilament; NF) (F). Scale bar = 100 µm. (G) RT-PCR of differentiation markers in undifferentiated iPS cell colony G (Undifferentiation) and embryoid bodies derived from iPS cell colony G. Differentiation). (H–K) Hematoxylin and eosin staining of teratoma formed by transplantation of iPS cell colony G into immunodeficient mice testis. (H), Low magnification of the formed teratoma (12 weeks after injection). Endodermal (I), mesodermal (J) and ectodermal (K) tissue were observed in the teratoma.</p

Classification of morphologic characteristics of colonies generated from human fibroblasts using four retroviral vectors encoding Oct3/4, Sox2, Klf4, and c-Myc.

<p>Each replicate represented 2×10⁵ transduced cells seeded onto a 60-mm dish containing feeder cells and cultured in Primate ES cell medium for thirty days.</p

Effects of cell therapy with vehicle, BMSCs, non-Muse cells, and Muse cells on memory impairment at 7 and 35 days after permanent middle cerebral artery occlusion (pMCAO).

<p>Correct choices (A) and errors (B) in the eight-arm radial maze task. **, ††, ¶¶ P<0.01 vs. vehicle-treated mice.</p

Morphometric analysis in colonies generated from human fibroblasts by using a single polycistronic Oct3/4-Klf4-Sox2-c-Myc-GFP expressing viral vector.

<p>(H–L, G) Photograph and parameters of colonies H∼L and G. Graphs shows parameters of each classified colony, including that of iPS cell colony G. The numerical value in the graph indicate the ratio to the maximum value (setting 100 for maximum value) in each parameter. Scale bars = 100 µm.</p

Representative coronal sections of infarct area on triphenyltetrazolium chloride (TTC) staining at 24 hr after permanent middle cerebral artery occlusion.

<p>Infarct volume was measured as 22.9 ± 2.9% of the contralateral hemisphere. Scale bar = 5 mm.</p