Efficient Generation of Non-Integration and Feeder-Free Induced Pluripotent Stem Cells from Human Peripheral Blood Cells by Sendai Virus

Background/Aims: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. Methods: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. Results: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. Conclusion: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications

A Bi-Functional Anti-Thrombosis Protein Containing Both Direct-Acting Fibrin(ogen)olytic and Plasminogen-Activating Activities

Direct-acting fibrin(ogen)olytic agents such as plasmin have been proved to contain effective and safety thrombolytic potential. Unfortunately, plasmin is ineffective when administered by the intravenous route because it was neutralized by plasma antiplasmin. Direct-acting fibrin(ogen)olytic agents with resistance against antiplasmin will brighten the prospect of anti-thrombosis. As reported in ‘Compendium of Materia Medica’, the insect of Eupolyphaga sinensis Walker has been used as traditional anti-thrombosis medicine without bleeding risk for several hundreds years. Currently, we have identified a fibrin(ogen)olytic protein (Eupolytin1) containing both fibrin(ogen)olytic and plasminogen-activating (PA) activities from the beetle, E. sinensis. Objectives: To investigate the role of native and recombinant eupolytin1 in fibrin(ogen)olytic and plasminogen-activating processes. Methods and Results: Using thrombus animal model, eupolytin1 was proved to contain strong and rapid thrombolytic ability and safety in vivo, which are better than that of urokinase. Most importantly, no bleeding complications were appeared even the intravenous dose up to 0.12 µmol/kg body weight (3 times of tested dose which could completely lyse experimental thrombi) in rabbits. It is the first report of thrombolytic agents containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. Conclusions: The study identified novel thrombolytic agent with prospecting clinical potential because of its bi-functional merits containing both plasmin- and PA-like activities and unique pharmacological kinetics in vivo

Signature microRNA expression profile is associated with spontaneous hypertension in African green monkey

Chlorocebus aethiops sabaeus, the African Green monkey (AGM), has been proved to exhibit renal vascular remodeling and spontaneous hypertension. However, little is known about the roles of microRNAs (miRNAs) in this process.Using small RNA deep sequencing, we compared the plasma miRNA expression patterns between hypertensive (HT) AGMs and normotensive (NT) AGMs. Expression of miRNAs (miR-122, miR-339, miR-296-5p) was validated independently in plasma samples from 10 HT AGMs and 10 NT AGMs (fold changes are 2.0, 1.6, 2.7 respectively; all P< 0.001). Potential BP (blood pressure)-regulating mRNA targets were predicted by TargetScan and confirmed in the Vero cells. We report for the first time a circulating miRNA profile for AGM. miRNAs, such as miR-122, miR-339, miR-296-5p, may be involved in renal pathologies and spontaneous hypertension of AGM

Hospital No.307 of PLA, the

receptor – an in silico analysis approac

iRhom2 Mutation Leads to Aberrant Hair Follicle Differentiation in Mice

<div><p>iRhom1 and iRhom2 are inactive homologues of rhomboid intramembrane serine proteases lacking essential catalytic residues, which are necessary for the maturation of TNFα-converting enzyme (TACE). In addition, iRhoms regulate epidermal growth factor family secretion. The functional significance of iRhom2 during mammalian development is largely unclear. We have identified a spontaneous single gene deletion mutation of <i>iRhom2</i> in <i>Uncv</i> mice. The <i>iRhom2^Uncv/Uncv</i> mice exhibit hairless phenotype in a BALB/c genetic background. In this study, we observed dysplasia hair follicles in <i>iRhom2^Uncv/Uncv</i> mice from postnatal day 3. Further examination found decreased hair matrix proliferation and aberrant hair shaft and inner root sheath differentiation in <i>iRhom2^Uncv/Uncv</i> mutant hair follicles. iRhom2 is required for the maturation of TACE. Our data demonstrate that iRhom2<i>^Uncv</i> cannot induce the maturation of TACE in vitro and the level of mature TACE is also significantly reduced in the skin of <i>iRhom2^Uncv/Uncv</i> mice. The activation of Notch1, a substrate of TACE, is disturbed, associated with dramatically down-regulation of Lef1 in <i>iRhom2^Uncv/Uncv</i> hair follicle matrix. This study identifies iRhom2 as a novel regulator of hair shaft and inner root sheath differentiation.</p></div

Dysplasia hair follicles in <i>iRhom2^Uncv/Uncv</i> mice.

<p>(A–J) Histology of the dorsal skin from wild-type and <i>iRhom2^Uncv/Uncv</i> mice at E15.5, E17.5, P0, P3 and P9, respectively. (K, L) TUNEL staining in the dorsal skin of wild-type and <i>iRhom2^Uncv/Uncv</i> mice at P9. (M–R) Immunofluorescence staining of Ki67 in the dorsal skin of wild-type and <i>iRhom2^Uncv/Uncv</i> mice at P0, P3 and P9, respectively. (S) Number of hair follicles per mm at E17.5; n = 6. (T) Number of Ki67-positive cells per hair follicle matrix at P0, P3 and P9; n = 8, **P<0.01. WT, wildtype; HO, homozygous. Stars indicate the hair placodes. Scale bars: (A–F, M, N), 25 µm; (K, L), 12.5 µm; (G, H, O–R), 50 µm; (I–J), 100 µm.</p

Expression levels of regulators of inner root sheath and hair shaft differentiation in <i>iRhom2^Uncv/Uncv</i> follicles.

<p>(A, B) Immunofluorescence staining of NICD in the dorsal skin of P9 wild-type and age-matched <i>iRhom2^Uncv/Uncv</i> mice. (C–J) Immunohistochemistry for Lef1 in the dorsal skin of E17.5, P0, P3 and P9 wild-type and <i>iRhom2^Uncv/Uncv</i> mice. (K) Western blot analysis of NICD protein expression in the dorsal skin of wild-type and <i>iRhom2^Uncv/Uncv</i> mice at P0, P3, P5 and P9. (L) Notch signaling transcriptional activity affected by Flag-iRhom2 or Flag-iRhom2<i>^Uncv</i> mutation in HeLa cells. NICD serves as a positive control. (M) Western blot analysis of Lef1 protein expression in the dorsal skin of wild-type and <i>iRhom2^Uncv/Uncv</i> mice at P0, P2, P3, P5 and P9. (N) Real-time PCR analysis of <i>Notch1</i>, <i>Hes1</i>, <i>Hey1</i>, <i>Hey2</i>, <i>Bmp2</i>, <i>Bmp4</i>, <i>Lef1</i>, <i>Foxn1</i>, <i>Gata3</i>, <i>Ctnnb1</i>, <i>Tcf3</i>, <i>Dact1</i>, <i>Wif1</i> and <i>Daam2</i> mRNA expression in the dorsal skin of wild-type and <i>iRhom2^Uncv/Uncv</i> mice at P5. n = 3, *P<0.05, **P<0.01. Scale bars: (A, B), 12.5 µm; (C, D), 50 µm; (E–J), 25 µm.</p