lineage-reprogramming-pericyte-derived-cells-adult-human-brain-into

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Ludwig Maximilians University Munich

Human mesenchymal stem cells (hMSCs) represent promising tools in various clinical applications, including the regeneration of injured tissues by endogenous or transplanted hMSCs. The molecular mechanisms, however, that control hMSC mobilization and homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. We have analyzed bone marrow-derivedhMSCs and detected strong expression and synthesis of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. The ability of hMSCs to traverse reconstituted human basement membranes was effectively blocked in the presence of synthetic MMP inhibitors. Detailed studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired hMSC invasion, whereas silencing of TIMP-1 enhanced cell migration, indicating opposing roles of both TIMPs in this process. Moreover, the inflammatory cytokines TGF-beta1, IL-1beta, and TNF-alpha up-regulated MMP-2, MT1-MMP, and/or MMP-9 production in these cells, resulting in a strong stimulation of chemotactic migration through ECM, whereas the chemokine SDF-1alpha exhibited minor effects on MMP/TIMP expression and cell invasion. Thus, induction of specific MMP activity in hMSCs by inflammatory cytokines promotes directed cell migration across reconstituted basement membranes in vitro providing a potential mechanism in hMSC recruitment and extravasation into injured tissues in vivo.

MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines.

The regulative network conducting adult stem cells in endogenous tissue repair is of prime interest for understanding organ regeneration as well as preventing degenerative and malignant diseases. One major signal transduction pathway which is involved in the control of these (patho)physiological processes is the Wnt pathway. Recent results obtained in our laboratories showed for the first time that canonical Wnt signaling is critically involved in the control of the migration/invasion behaviour of human mesenchymal stem cells (hMSC). In the first part of this review, we describe that the regenerative state is closely linked to the activation of the Wnt pathway. Central hallmarks of activated stem cells are recapitulated in a similar way also in cancer metastasis, where the acquisition of an invasive cancer stem cell phenotype is associated with the induction of Wnt-mediated epithelial to mesenchymal transition (EMT). In the second part, the influence of proinflammatory cytokines such as transforming growth factor (TGF-)beta1, interleukin (Il-)1beta, and tumor necrosis factor (TNF-)alpha is discussed with regard to the invasive characteristics of hMSC. In this context, special attention has been paid on the role of matrix metalloproteinases (MMPs), such as MMP-2, MMP-9 and membrane type 1 (MT1)-MMP, as well as on the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. Putative cross-talks between different signal transduction pathways that may amplify the invasive capacity of this stem cell population are also discussed. Finally, the consequences towards future drug-mediated therapeutical modifications of Wnt signaling in stem cells and tumor cells are highlighted.

The Wnt signal transduction pathway in stem cells and cancer cells: influence on cellular invasion.

The capacity of human mesenchymal stem cells (hMSC) for self-renewal and differentiation is a tightly regulated process within their microenvironment--the stem cell niche. For future therapeutic applications of hMSC within the frame of tissue engineering, it is of major importance to understand the factors involved in triggering differentiation cascades of hMSC. Using either osteoblast-conditioned medium or an indirect coculture system, we investigated whether soluble factors from human osteoblasts (hOB) are sufficient to induce early osteogenic markers in hMSC. Thereby, we detected an induction of several osteogenic markers like alkaline phosphatase, bone sialoprotein 2, leptin receptor, decorin, and cathepsin K in hMSC as indicators of the onset of early osteogenesis. Further, because Wnt signaling has been reported to play an important role in osteogenesis, we performed RNAi against the main Wnt mediator beta-catenin and the low-density lipoprotein receptor-related protein 5 as a major Wnt co-receptor in hMSC. Whereas alkaline phosphatase was significantly downregulated with this approach, the other osteogenic markers showed a markedly upregulation. These observations suggest that hOB-secreted factors could induce early osteogenic markers in hMSC. Thus, with regard to a therapeutic setting, these findings may pave the way for a more in vivo-related differentiation procedure for the generation of osteoblast-like cells.

Human osteoblast-derived factors induce early osteogenic markers in human mesenchymal stem cells.

Based on the capacity of mesenchymal stem cells (MSC) to differentiate into multiple cell types in vitro and in vivo, MCS may be a suitable source for cell therapy and regeneration strategies. A prerequisite for effective clinical applications of human MSC (hMSC) is a profound knowledge of signal transduction cascades that mediate processes like proliferation, targeted migration and differentiation. Recently, we identified the canonical Wnt signal transduction pathway as a key player in hMSC proliferation and invasion. To evaluate whether those findings are transferable to the equivalent counterparts in mice, we studied important steps in the wingless/int-1 (Wnt) signal transduction pathway in mouse MSC (mMSC) and mMSC carrying a T cell specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF)-reporter transgene. We found that the induction of the canonical Wnt pathway resulted in the up-regulation of the known Wnt target gene cyclin D1, closely associated with an enhanced proliferation capacity of mMSC. Interestingly, the expression of the Wnt target gene membrane type 1-matrix metalloproteinase (MT1-MMP) was diminished in mMSC upon Wnt3a stimulation, which came along with an impaired invasion. In line with these findings, MMP-2 and MMP-9 expression levels in mMSC were also decreased after Wnt3a treatment. In contrast, inhibition of Wnt signalling by the knockdown of the transcriptional activator beta-catenin resulted in an up-regulation of MT1-MMP and mMSC invasion. By comparing these findings with the settings in hMSC, major differences in Wnt-regulated MMP expression were observed in mMSC. Thus, our data advice caution when mouse model systems represent the pre-clinical validation of MSC-mediated therapeutical approaches.

Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression.

The φC31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells.

The therapeutic potential of ΦC31 integrase as a gene therapy system.

Supplementary to the efficient inhibition of trypsin, chymotrypsin, plasma kallikrein, and plasmin already described by the EcTI inhibitor from Enterolobium contortisiliquum, it also blocks human neutrophil elastase (K(iapp)=4.3 nM) and prevents phorbol ester (PMA)-stimulated activation of matrix metalloproteinase (MMP)-2 probably via interference with membrane-type 1 (MT1)-MMP. Moreover, plasminogen-induced activation of proMMP-9 and processing of active MMP-2 was also inhibited. Furthermore, the effect of EcTI on the human cancer cell lines HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), K562 and THP-1 (leukemia), as well as on human primary fibroblasts and human mesenchymal stem cells (hMSCs) was studied. EcTI inhibited in a concentration range of 1.0-2.5 μM rather specifically tumor cell viability without targeting primary fibroblasts and hMSCs. Taken together, our data indicate that the polyspecific proteinase inhibitor EcTI prevents proMMP activation and is cytotoxic against tumor cells without affecting normal tissue remodeling fibroblasts or regenerative hMSCs being an important tool in the studies of tumor cell development and dissemination.

The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines.

WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the β-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced β-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the β-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.

Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways.

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Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

Cellules souches pluripotentes induites (CISP) ont r&#xE9;volutionn&#xE9; le domaine des cellules souches. CSPi sont le plus souvent fabriqu&#xE9;s en utilisant des r&#xE9;trovirus. Cependant, les cellules r&#xE9;sultantes peuvent &#xEA;tre mal adapt&#xE9;s pour des applications cliniques. Beaucoup de strat&#xE9;gies alternatives pour faire CSPi ont &#xE9;t&#xE9; d&#xE9;velopp&#xE9;s, mais les strat&#xE9;gies nonintegrating tendance &#xE0; &#xEA;tre inefficaces, tandis que les strat&#xE9;gies d&#x26;#39;int&#xE9;gration impliquent l&#x26;#39;int&#xE9;gration al&#xE9;atoire. Ici, nous rapportons une strat&#xE9;gie facile &#xE0; cr&#xE9;er CSPi murins qui utilise l&#x26;#39;ADN plasmidique et la transfection unique avec une s&#xE9;quence-sp&#xE9;cifiques recombinases. PhiC31 int&#xE9;grase a &#xE9;t&#xE9; utilis&#xE9; pour ins&#xE9;rer la cassette dans le g&#xE9;nome de reprogrammation, la production de CSPi. Recombinase Cre a ensuite &#xE9;t&#xE9; utilis&#xE9;e pour l&#x26;#39;excision des g&#xE8;nes de reprogrammation. Les CSPi ont &#xE9;t&#xE9; d&#xE9;montr&#xE9;es d&#x26;#39;&#xEA;tre pluripotentes par in vitro et in vivo, des crit&#xE8;res &#xE0; la fois avant et apr&#xE8;s l&#x26;#39;excision de la cassette reprogrammation. Cette strat&#xE9;gie est comparable avec les approches en mati&#xE8;re d&#x26;#39;efficacit&#xE9; r&#xE9;troviraux, mais est class&#xE9; non dangereux pour l&#x26;#39;utilisateur, simple &#xE0; r&#xE9;aliser, et les r&#xE9;sultats en mati&#xE8;re d&#x26;#39;int&#xE9;gration non al&#xE9;atoire d&#x26;#39;une cassette reprogrammation qui peut &#xEA;tre facilement supprim&#xE9;. Nous avons d&#xE9;montr&#xE9; l&#x26;#39;efficacit&#xE9; de cette strat&#xE9;gie reprogrammation et l&#x26;#39;excision dans les deux types de cellules accessibles, les fibroblastes et les cellules souches adipeuses. Cette strat&#xE9;gie simple produit des cellules souches pluripotentes qui ont le potentiel pour &#xEA;tre utilis&#xE9; dans un contexte clinique.

Site sp&#xE9;cifique Strat&#xE9;gie recombinase pour cr&#xE9;er cellules souches pluripotentes induites de mani&#xE8;re efficace avec l&#x26;#39;ADN plasmidique

Induzierten pluripotenten Stammzellen (iPSCs) haben die Stammzell-Bereich revolutioniert. iPSCs sind meist mit Retroviren erzeugt. Jedoch k&ouml;nnen die resultierenden Zellen f&uuml;r klinische Anwendungen ungeeignet sein. Viele alternative Strategien zu iPSCs entwickelt worden, aber die nonintegrating Strategien sind in der Regel als ineffizient erweisen, w&auml;hrend die integrierenden Strategien zuf&auml;llige Integration umfassen. Hier berichten wir eine einfache Strategie murinen iPSCs zu erstellen, die Plasmid-DNA und einzelne Transfektion mit Sequenz-spezifische Recombinases verwendet. PhiC31 Integrase wurde verwendet, um das Genom, produzieren iPSCs die Neuprogrammierung Kassette einlegen. CRE Recombinase wurde dann f&uuml;r Exzision der Neuprogrammierung Gene verwendet. Die iPSCs wurden pluripotente werden durch in-vitro- und in-vivo-Kriterien, demonstriert, sowohl vor als auch nach Exzision der Neuprogrammierung Kassette. Diese Strategie ist vergleichbar mit Retroviren Ans&auml;tzen in Effizienz, sondern ist f&uuml;r den Benutzer einfach zu erf&uuml;llen, ungef&auml;hrlichen und f&uuml;hrt gezielt Integration einer Neuprogrammierung Kassette, die ohne weiteres gel&ouml;scht werden k&ouml;nnen. Wir bewiesen die Effizienz dieser Strategie Neuprogrammierung und Exzision in zwei zug&auml;nglich Zelltypen, Fibroblasten und adip&ouml;se Stammzellen. Diese einfache Strategie produziert pluripotenten Stammzellen, die in einer klinischen Einstellung verwendet werden k&ouml;nnen.

Standortspezifische Recombinase Strategie induzierten pluripotenten Stammzellen effizient mit Plasmid DNA zu erstellen.

Le cellule staminali pluripotenti indotte (iPSCs) hanno rivoluzionato il campo delle cellule staminali. iPSCs pi&ugrave; spesso sono prodotte utilizzando retrovirus. Tuttavia, le cellule risultanti possono essere inadatte per applicazioni cliniche. Sono state sviluppate molte strategie alternative per fare iPSCs, ma le strategie di nonintegrating tendono ad essere inefficiente, mentre le strategie di integrazione coinvolgono integrazione casuale. Riportiamo una strategia facile creare iPSCs murino che utilizza il DNA del plasmide e transfezione singolo con sequenza specifica ricombinasi. Integrase PhiC31 &egrave; stato utilizzato per inserire la cassetta riprogrammazione del genoma, producendo iPSCs. CRE recombinase allora &egrave; stato utilizzato per l&#39;asportazione dei geni riprogrammazione. Le iPSCs sono state dimostrate essere pluripotenti da criteri in vitro e in vivo, sia prima che dopo l&#39;asportazione della cassetta riprogrammazione. Questa strategia &egrave; paragonabile con approcci retrovirali in efficienza, ma non pericolosa per l&#39;utente, semplice da eseguire e risultati casuali integrazione di una riprogrammazione cassetta che pu&ograve; essere facilmente eliminata. Abbiamo dimostrato l&#39;efficacia di questa strategia di riprogrammazione e l&#39;escissione in cella accessibile due tipi, fibroblasti e cellule staminali adipose. Questa semplice strategia produce cellule staminali pluripotenti, che hanno il potenziale per essere utilizzato in una regolazione clinica.

Strategia recombinase site-specific per creare cellule staminali pluripotenti indotte in modo efficiente con il DNA del plasmide.

&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#xFF08;&#x6027;IPSC&#xFF09;&#x306F;&#x3001;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5206;&#x91CE;&#x306B;&#x9769;&#x547D;&#x3092;&#x3082;&#x305F;&#x3089;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6027;IPSC&#x306F;&#x3001;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x5834;&#x5408;&#x3001;&#x30EC;&#x30C8;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x3092;&#x7528;&#x3044;&#x3066;&#x751F;&#x7523;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x5F97;&#x3089;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x306F;&#x81E8;&#x5E8A;&#x5FDC;&#x7528;&#x306E;&#x305F;&#x3081;&#x306B;&#x4E0D;&#x9069;&#x5F53;&#x304B;&#x3082;&#x3057;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;&#x6027;IPSC&#x3092;&#x3059;&#x308B;&#x591A;&#x304F;&#x306E;&#x4EE3;&#x66FF;&#x6226;&#x7565;&#x304C;&#x958B;&#x767A;&#x3055;&#x308C;&#x305F;&#x304C;&#x3001;&#x7D71;&#x5408;&#x6226;&#x7565;&#x304C;&#x30E9;&#x30F3;&#x30C0;&#x30E0;&#x306A;&#x7D71;&#x5408;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3057;&#x306A;&#x304C;&#x3089;nonintegrating&#x6226;&#x7565;&#x306F;&#x3001;&#x975E;&#x52B9;&#x7387;&#x306B;&#x306A;&#x308B;&#x50BE;&#x5411;&#x304C;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30C9;DNA&#x3068;&#x914D;&#x5217;&#x7279;&#x7570;&#x7684;&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC;&#x3092;&#x6301;&#x3064;&#x5358;&#x4E00;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x6027;IPSC&#x3092;&#x4F5C;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5BB9;&#x6613;&#x306A;&#x6226;&#x7565;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002; PhiC31&#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30E9;&#x30FC;&#x30BC;&#x306F;&#x3001;&#x6027;IPSC&#x3092;&#x751F;&#x7523;&#x3001;&#x30B2;&#x30CE;&#x30E0;&#x306B;&#x66F8;&#x304D;&#x63DB;&#x3048;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x633F;&#x5165;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002; Cre&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC;&#x306F;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5207;&#x9664;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002;&#x6027;IPSC&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x5207;&#x9664;&#x524D;&#x3068;&#x5F8C;&#x306E;&#x4E21;&#x65B9;&#x3067;&#x3001;in vitro&#x304A;&#x3088;&#x3073;in vivo&#x57FA;&#x6E96;&#x306E;&#x306B;&#x3088;&#x3063;&#x3066;&#x591A;&#x80FD;&#x6027;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x5B9F;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x6226;&#x7565;&#x306F;&#x3001;&#x52B9;&#x7387;&#x6027;&#x306E;&#x30EC;&#x30C8;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3068;&#x540C;&#x7B49;&#x3067;&#x3042;&#x308B;&#x304C;&#x3001;&#x5B9F;&#x884C;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x30E6;&#x30FC;&#x30B6;&#x30FC;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x5371;&#x967A;&#x6027;&#x306E;&#x306A;&#x3044;&#x5358;&#x7D14;&#x306A;&#x3082;&#x306E;&#x3067;&#x3001;&#x5BB9;&#x6613;&#x306B;&#x524A;&#x9664;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x306E;&#x30E9;&#x30F3;&#x30C0;&#x30E0;&#x3067;&#x7D71;&#x5408;&#x306E;&#x7D50;&#x679C;&#x3002;&#x6211;&#x3005;&#x306F;2&#x3064;&#x200B;&#x200B;&#x306E;&#x30A2;&#x30AF;&#x30BB;&#x30B9;&#x53EF;&#x80FD;&#x306A;&#x7A2E;&#x985E;&#x306E;&#x7D30;&#x80DE;&#x3001;&#x7DDA;&#x7DAD;&#x82BD;&#x7D30;&#x80DE;&#x3001;&#x8102;&#x80AA;&#x5E79;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x3001;&#x3053;&#x306E;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x3084;&#x5207;&#x9664;&#x6226;&#x7565;&#x306E;&#x52B9;&#x7387;&#x6027;&#x3092;&#x5B9F;&#x8A3C;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5358;&#x7D14;&#x306A;&#x6226;&#x7565;&#x306F;&#x3001;&#x81E8;&#x5E8A;&#x306E;&#x73FE;&#x5834;&#x3067;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x79D8;&#x3081;&#x3066;&#x3044;&#x308B;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x751F;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30D7;&#x30E9;&#x30B9;&#x30DF;&#x30C9;DNA&#x3092;&#x52B9;&#x7387;&#x7684;&#x306B;&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x3092;&#x4F5C;&#x6210;&#x3059;&#x308B;&#x90E8;&#x4F4D;&#x7279;&#x7570;&#x7684;&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC;&#x6226;&#x7565;

&#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; (iPSCs) &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xBD84;&#xC57C;&#xB97C; &#xD601;&#xBA85;&#xC744; &#xC77C;&#xC73C;&#xCF30;&#xB2E4;. Ipscs&#xB294; &#xAC00;&#xC7A5; &#xC790;&#xC8FC; retroviruses&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC0DD;&#xC0B0; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xADF8; &#xACB0;&#xACFC; &#xC138;&#xD3EC; &#xBCD1; &#xC784;&#xC0C1; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0; &#xC801;&#xD569; &#xC788;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. IPSCs &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xB9CE;&#xC740; &#xB300;&#xCCB4; &#xC804;&#xB7B5;&#xC744; &#xAC1C;&#xBC1C; &#xD558; &#xACE0;, &#xADF8;&#xB7EC;&#xB098; nonintegrating &#xC804;&#xB7B5; &#xD1B5;&#xD569; &#xC804;&#xB7B5; &#xC784;&#xC758;&#xC758; &#xD1B5;&#xD569;&#xC744; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xC218; &#xACBD;&#xD5A5;&#xC774; &#xC788;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xBCF4;&#xACE0; murine iPSCs &#xD50C;&#xB77C;&#xC2A4; &#xBBF8;&#xB4DC; DNA &#xBC0F; &#xB2E8;&#xC77C; transfection &#xC2DC;&#xD000;&#xC2A4; &#xD2B9;&#xC815; recombinases&#xC640; &#xD568;&#xAED8; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB9CC;&#xB4DC;&#xB294; &#xC190;&#xC26C;&#xC6B4; &#xC804;&#xB7B5;. PhiC31 integrase iPSCs &#xC0DD;&#xC0B0; &#xAC8C;&#xB188; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xCE74;&#xC138;&#xD2B8; &#xC0BD;&#xC785; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9;&#xD55C;. Cre recombinase&#xB294; &#xB2E4;&#xC74C; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC720;&#xC804;&#xC790;&#xC758; &#xC808;&#xB2E8;&#xC5D0; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. IPSCs &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xCE74;&#xC138;&#xD2B8;&#xC758; &#xC808;&#xB2E8; &#xC804;&#xD6C4;&#xC5D0; &#xBAA8;&#xB450; &#xC0DD;&#xCCB4; &#xC678; &#xBC0F; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xAE30;&#xC900;&#xC5D0; &#xC758;&#xD574; &#xC218; pluripotent &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. &#xC774; &#xC804;&#xB7B5; &#xD6A8;&#xC728;, retroviral &#xC811;&#xADFC;&#xBC95;&#xACFC; &#xBE44;&#xAD50; &#xC774;&#xC9C0;&#xB9CC; nonhazardous &#xC0AC;&#xC6A9;&#xC790;, &#xC218;&#xD589;, &#xAC04;&#xB2E8; &#xD558; &#xACE0; &#xC27D;&#xAC8C; &#xC0AD;&#xC81C;&#xD560; &#xC218; &#xC788;&#xB294; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xCE74;&#xC138;&#xD2B8;&#xC758; nonrandom &#xD1B5;&#xD569;&#xC5D0;&#xC11C; &#xACB0;&#xACFC;. &#xC6B0;&#xB9AC; &#xB450; &#xC561;&#xC138;&#xC2A4;&#xD560; &#xC218; &#xC140; &#xC720;&#xD615;&#xACFC; &#xC12C;&#xC720; &#xC544; &#xC138;&#xD3EC;, &#xC9C0;&#xBC29; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; &#xBC0F; &#xC808;&#xB2E8;&#xC774; &#xC804;&#xB7B5;&#xC758; &#xD6A8;&#xC728;&#xC131;&#xC744; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. &#xC774; &#xAC04;&#xB2E8;&#xD55C; &#xC804;&#xB7B5; &#xC784;&#xC0C1; &#xC124;&#xC815;&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC0DD;&#xC131;.

&#xC0AC;&#xC774;&#xD2B8;&#xBCC4; recombinase &#xD50C;&#xB77C;&#xC2A4; &#xBBF8;&#xB4DC; DNA&#xC640; &#xD568;&#xAED8; &#xD6A8;&#xC728;&#xC801;&#xC73C;&#xB85C; &#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xB97C; &#xB9CC;&#xB4DC;&#xB294; &#xC804;&#xB7B5;.

C&#xE9;lulas madre pluripotentes inducidas (iPSCs) han revolucionado el campo de c&#xE9;lulas madre. iPSCs son m&#xE1;s a menudo producida mediante el uso de los retrovirus. Sin embargo, las c&#xE9;lulas resultantes pueden ser poco adecuados para aplicaciones cl&#xED;nicas. Muchas de las estrategias alternativas de hacer iPSCs se han desarrollado, pero las estrategias nonintegrating tienden a ser ineficientes, mientras que las estrategias de integraci&#xF3;n implica la integraci&#xF3;n al azar. Aqu&#xED;, se presenta una estrategia f&#xE1;cil para crear iPSCs murinos que utiliza el ADN pl&#xE1;smido y transfecci&#xF3;n individual con secuencias espec&#xED;ficas de recombinaciones. PhiC31 integrasa se utiliza para insertar el casete reprogramaci&#xF3;n en el genoma, produciendo iPSCs. Recombinasa Cre se utiliz&#xF3; para la escisi&#xF3;n de los genes de reprogramaci&#xF3;n. Los CMPi demostraron ser pluripotentes por in vitro e in vivo en los criterios, tanto antes como despu&#xE9;s de la escisi&#xF3;n de la casete de reprogramaci&#xF3;n. Esta estrategia es comparable con los enfoques retrovirales en la eficiencia, pero no sea peligrosa para el usuario, f&#xE1;cil de realizar, y da como resultado la integraci&#xF3;n aleatoria de una casete de reprogramaci&#xF3;n que puede ser f&#xE1;cilmente eliminada. Hemos demostrado la eficacia de esta estrategia de reprogramaci&#xF3;n y la escisi&#xF3;n en dos tipos de c&#xE9;lulas accesibles, fibroblastos y c&#xE9;lulas adiposas madre. Esta estrategia simple produce c&#xE9;lulas madre pluripotentes que tienen el potencial para ser utilizado en un entorno cl&#xED;nico.

Sitio espec&#xED;fico de la Estrategia recombinasa para crear c&#xE9;lulas madre pluripotentes inducidas de manera eficiente con el ADN del pl&#xE1;smido

Human mesenchymal stem cells (hMSC) are subjected to the control of several signal transduction pathways during regeneration processes, whereby Wnt/β-catenin signaling is of pivotal importance. Since there exists only fragmentary knowledge concerning the molecular function of the Wnt-coreceptors LRP5 and LRP6 (low-density lipoprotein receptor-related protein) in hMSC, we studied their impact on Wnt/β-catenin signal transduction by RNA interference. For monitoring changes in β-catenin-dependent transcription in a highly sensitive and specific manner, hMSC were stably transfected with a TCF/LEF reporter gene plasmid. In the presence of the activator Wnt3a, knockdown of LRP6 led to a strong decreased Wnt/β-catenin signaling, while RNAi against LRP5 exhibited no effect in this setting. In a reverse approach, ectopic expression of LRP6 resulted in a strong enhancement of Wnt/β-catenin signaling, whereas overexpression of LRP5 exhibited no increased signaling capacity. Furthermore, only the ectopic expression of LRP6--but not that of LRP5--was able to restore Wnt3a-mediated β-catenin signaling after knockdown of endogenously expressed LRP6. These results demonstrate LRP6 as the predominant Wnt3a LRP-receptor in hMSC, which cannot be substituted by LRP5. In addition, we observed enhanced differentiation toward the adipogenic lineage after RNAi against LRP6 which was associated with the induction of PPAR-γ and fat vacuole formation. Thus, LRP6 is not only indispensable for Wnt3a/β-catenin signaling, but also for the suppression of differentiation of hMSC into the adipogenic lineage. Based on these observations, LRP6 may represent an attractive drug target for manipulating hMSC in cell and tissue regeneration approaches.

LRP6 mediates Wnt/β-catenin signaling and regulates adipogenic differentiation in human mesenchymal stem cells.

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. A major challenge for the translation of neuronal reprogramming into therapy is whether the adult human brain contains cell populations amenable to direct somatic cell conversion. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks can be reprogrammed into neuronal cells by retrovirus-mediated coexpression of the transcription factors Sox2 and Mash1. These induced neuronal cells acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons, indicating their capability of integrating into neural networks. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue-nonspecific alkaline phosphatase promoter corroborated the pericytic origin of the reprogrammed cells. Our results raise the possibility of functional conversion of endogenous cells in the adult human brain to induced neuronal fates.

Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells.

Pericytes, typically attached to the walls of microvessels in almost all organs, interact with endothelial cells and take part in diverse biological processes, e.g. blood vessel regulation and tissue repair. This suggests that pericytes harbor a remarkable degree of cellular plasticity, which could potentially be employed for the treatment of diseases affecting diverse tissues such as the skeletal muscle and the central nervous system. Here, we follow pericytes on their journey across Waddington's epigenetic landscape, descending from their origin, along a path guided by environmental signals or ectopic transcription factors, at the end of which they acquire a new identity, e.g. muscle or nerve cells. The central theme of this review is the question of whether pericytes can be enticed to differentiate into whatever cell type is needed, and thus provide an endogenous cellular source for treating as yet incurable diseases--like a magic bullet.

Mountaineering pericytes--a universal key to tissue repair?

A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

Article	Year
MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines. Blood\| PubMed ID: 17197427	2007
The Wnt signal transduction pathway in stem cells and cancer cells: influence on cellular invasion. Stem cell reviews\| PubMed ID: 17873378	2007
Human osteoblast-derived factors induce early osteogenic markers in human mesenchymal stem cells. Tissue engineering. Part A\| PubMed ID: 19292682	2009
Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression. Journal of cellular and molecular medicine\| PubMed ID: 19413884	2009
The therapeutic potential of ΦC31 integrase as a gene therapy system. Expert opinion on biological therapy\| PubMed ID: 21736536	2011
The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines. Biological chemistry\| PubMed ID: 21781023	2011
Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways. Biological chemistry\| PubMed ID: 21864196	2011
Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA. Stem cells (Dayton, Ohio)\| PubMed ID: 21898697	2011
LRP6 mediates Wnt/β-catenin signaling and regulates adipogenic differentiation in human mesenchymal stem cells. The international journal of biochemistry & cell biology\| PubMed ID: 22871567	2012
Safe genetic modification of cardiac stem cells using a site-specific integration technique. Circulation\| PubMed ID: 22965984	2012
Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells. Cell stem cell\| PubMed ID: 23040476	2012
Mountaineering pericytes--a universal key to tissue repair? BioEssays : news and reviews in molecular, cellular and developmental biology\| PubMed ID: 23757054	2013
Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells. PloS one\| PubMed ID: 24781921	2014

Marisa Karow

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