alternative-cultures-for-human-pluripotent-stem-cell-production

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

National Institutes of Health

Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ. EGFP(+)/NG2(+) cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.

Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development.

The current studies focus on what mechanisms regulate the concentration of PLP mRNA in cells. The PLP mRNA is very stable and these studies suggest that its stability is regulated by a trans-acting factor specific to oligodendrocytes. In order to test whether the 3'untranslated region (3'UTR) of the PLP mRNA might regulate PLP RNA stability, C6 cells were transfected with cDNAs that expressed either luciferase or luciferase fused to the 3'UTR of PLP. Although transgene expression was low, in cells transfected with the PLP 3'UTR, there was a significant decrease in the endogenous PLP mRNA. These cells showed a distinct change in morphology and in adhesion properties. Thus, there may be a role for plp gene products in cell adhesion, which was downregulated in these cells, or an unknown function may be encoded by the PLP 3'UTR. Transgenic mice that overexpress enhanced green fluorescent protein fused to the PLP 3'UTR under control of PLP regulatory sequences were tested for the expression of the endogenous PLP mRNA. Three of four lines of transgenic mice had decreased endogenous PLP mRNA, relative to their non-transgenic littermates; the EGFP-PLP 3'UTR mouse line that expressed the highest level of transgene mRNA had a 54% reduction in PLP mRNA. We hypothesize that the PLP mRNA is regulated by elements in the 3'UTR and stabilizing proteins specific to oligodendrocytes, and that in cells that overexpress the PLP 3'UTR, these stabilizing proteins may be insufficient to maintain the normal level of the endogenous PLP mRNA.

Overexpression of the 3'-untranslated region of myelin proteolipid protein mRNA leads to reduced expression of endogenous proteolipid mRNA.

Valproate, an anticonvulsant drug used to treat bipolar disorder, was studied for its ability to promote neurogenesis from embryonic rat cortical or striatal primordial stem cells. Six days of valproate exposure increased by up to fivefold the number and percentage of tubulin beta III-immunopositive neurons, increased neurite outgrowth, and decreased by fivefold the number of astrocytes without changing the number of cells. Valproate also promoted neuronal differentiation in human fetal forebrain stem cell cultures. The neurogenic effects of valproate on rat stem cells exceeded those obtained with the neurotrophins brain-derived growth factor (BDNF) or NT-3, and slightly exceeded the effects obtained with another mood stabilizer, lithium. No effect was observed with carbamazepine. Most of the newly formed neurons were GABAergic, as shown by 10-fold increases in neurons that immunostained for GABA and the GABA-synthesizing enzyme GAD65/67. Double immunostaining for bromodeoxyuridine and tubulin beta III showed that valproate increased by four- to fivefold the proliferation of neuronal progenitors derived from rat stem cells and increased cyclin D2 expression. Valproate also regulated the expression of survival genes, Bad and Bcl-2, at different times of treatment. The expression of prostaglandin E synthase, analyzed by quantitative RT-PCR, was increased by ninefold as early as 6 h into treatment by valproate. The enhancement of GABAergic neuron numbers, neurite outgrowth, and phenotypic expression via increases in the neuronal differentiation of neural stem cell may contribute to the therapeutic effects of valproate in the treatment of bipolar disorder.

The mood stabilizer valproic acid stimulates GABA neurogenesis from rat forebrain stem cells.

The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Toward xeno-free culture of human embryonic stem cells.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.

Non-colony type monolayer culture of human embryonic stem cells.

&#x5F88;&#x591A;&#x7531;&#x8BF1;&#x5BFC;&#x7684;&#x591A;&#x529F;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x6280;&#x672F;&#x751F;&#x6210;&#x7684;&#x5174;&#x594B;&#x4E0E;&#x5EFA;&#x6A21;&#x4EE5;&#x53CA;&#x6F5C;&#x5728;&#x7684;&#x4E2A;&#x6027;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x7684;&#x75BE;&#x75C5;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x6709;&#x5173;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8981;&#x8FFD;&#x6C42;&#x8FD9;&#x662F;&#x91CD;&#x8981;&#x7684;&#x662F;&#x8981;&#x4E86;&#x89E3;&#x5305;&#x62EC;&#x5176;&#x56FA;&#x6709;&#x53D8;&#x5F02;&#x6027;&#x591A;&#x80FD;&#x5E72;&#x6B63;&#x5E38;&#x72B6;&#x6001;&#x3002;&#x6211;&#x4EEC;&#x6267;&#x884C;&#x5404;&#x79CD;&#x5206;&#x5B50;&#x5206;&#x6790;&#x5316;&#x9A8C; 21 hESC &#x7EBF;&#x6761;&#x548C; 8 hiPSC &#x884C;&#x6765;&#x751F;&#x6210;&#x5168;&#x9762;&#x7684;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x672A;&#x5206;&#x5316;&#x72B6;&#x6001;&#x7684;&#x5FEB;&#x7167;&#x3002;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6570;&#x636E;&#x7684;&#x5206;&#x6790;&#x663E;&#x793A;&#xFF0C;&#x6839;&#x636E;&#x524D;&#x51E0;&#x6B21;&#x62A5;&#x544A;&#x6CA1;&#x6709;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7279;&#x5F02;&#x6027;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x3002;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x6BD4;&#x8F83;&#x5206;&#x5316;&#x4F5C;&#x4E3A;&#x80DA;&#x4F53; 2 &#x5A92;&#x4F53;&#x62DF;&#x542F;&#x52A8;&#x5BF9;&#x5355;&#x72EC;&#x7684;&#x5355;&#x5143;&#x683C;&#x7684;&#x547D;&#x8FD0;&#xFF0C;&#x4EE5;&#x53CA; 20 &#x6210;&#x4EBA;&#x7EC4;&#x7EC7;&#x5206;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x3002;&#x4ECE;&#x8FD9;&#x4E00;&#x5206;&#x6790;&#x6211;&#x4EEC;&#x5DF2;&#x7ECF;&#x4EA7;&#x751F;&#x4E86;&#x57FA;&#x56E0;&#x5217;&#x8868;&#x5B9A;&#x4E49;&#x591A;&#x6F5C;&#x80FD;&#xFF0C;&#x4E3A;&#x591A;&#x80FD;&#x5E72;&#x56FD;&#x5BB6;&#x5EFA;&#x7ACB;&#x4E00;&#x4E2A;&#x57FA;&#x7EBF;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x4F9B;&#x5217;&#x8868;&#x663E;&#x793A;&#x72EC;&#x7ACB;&#x5355;&#x5143;&#x683C;&#x547D;&#x8FD0;&#x7684;&#x62DF;&#x8BAE;&#x7684;&#x504F;&#x89C1;&#x8FD9;&#x4E24;&#x79CD;&#x5206;&#x5316;&#x6761;&#x4EF6;&#x4E30;&#x5BCC;&#x7684;&#x57FA;&#x56E0;&#x3002;

StemCellDB&#xFF1A; &#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x6570;&#x636E;&#x5E93;&#x5728;&#x56FD;&#x5BB6;&#x5065;&#x5EB7;&#x7814;&#x7A76;&#x6240;&#x3002;

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the 'normal' pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health.

Une grande partie de l&#39;enthousiasme g&eacute;n&eacute;r&eacute; par la technologie des cellules souches pluripotentes induites est pr&eacute;occup&eacute;e par la possibilit&eacute; de maladie de mod&eacute;lisation ainsi que le potentiel de la th&eacute;rapie cellulaire personnalis&eacute;. Toutefois, afin de poursuivre ce, il est important de comprendre l&#39;&eacute;tat de pluripotentes &laquo; normal &raquo;, y compris sa variabilit&eacute; inh&eacute;rente. Nous avons effectu&eacute; plusieurs essais mol&eacute;culaires de profilage pour 21 lign&eacute;es de CSEh et 8 lignes de hiPSC pour g&eacute;n&eacute;rer un aper&ccedil;u complet de l&#39;&eacute;tat indiff&eacute;renci&eacute; de cellules souches pluripotentes. Analyse les donn&eacute;es d&#39;expression g&eacute;nique a r&eacute;v&eacute;l&eacute; aucun mod&egrave;le d&#39;expression sp&eacute;cifiques iPSC g&egrave;ne conform&eacute;ment aux pr&eacute;c&eacute;dents rapports. Plus loin, nous avons compar&eacute; les cellules diff&eacute;renci&eacute;es comme corps embryo&iuml;des dans 2 m&eacute;dias propos&eacute;s de lancer la diff&eacute;renciation vers des destins cellulaires distincts, ainsi que 20 tissus adultes. De cette analyse, nous avons g&eacute;n&eacute;r&eacute; une liste de g&egrave;ne qui d&eacute;finit la pluripotence et &eacute;tablit une ligne de base pour l&#39;&eacute;tat pluripotent. Enfin, nous fournissons des listes de g&egrave;nes enrichis dans les deux conditions de diff&eacute;renciation qui montrent le biais propos&eacute; vers les destins de cellules ind&eacute;pendantes.

StemCellDB : La base de donn&eacute;es de cellules souches pluripotentes humaines au niveau National Institutes of Health.

Viel von der Aufregung von induzierten pluripotenten Stammzellen-Technologie befasst sich mit der M&ouml;glichkeit der Modellierung sowie das Potenzial f&uuml;r personalisierte Zelltherapie-Krankheit. Um dies zu verfolgen ist es jedoch wichtig zu verstehen, den &#39;normalen&#39; pluripotenten Zustand einschlie&szlig;lich ihrer inh&auml;renten Variabilit&auml;t. Wir haben verschiedene Molekulare Profilerstellungs-Assays f&uuml;r 21 hESC-Linien und 8 HiPSC Zeilen generieren einen umfassenden Snapshot des undifferenzierten pluripotenten Stammzellen durchgef&uuml;hrt. Analyse der Gen-Ausdruck-Daten ergab kein iPSC-spezifische Gen-Ausdruck-Muster nach fr&uuml;heren Berichten. Wir verglichen weitere Zellen, differenzierte als Embryoid K&ouml;rper in 2 Medien vorgeschlagene Differenzierung gegen&uuml;ber separate Zelle Schicksale sowie 20 Erwachsene Gewebe zu initiieren. Aus dieser Analyse haben wir eine gen-Liste erzeugt, die Pluripotenz definiert und etabliert eine Baseline f&uuml;r den pluripotenten Zustand. Schlie&szlig;lich bieten wir Listen von Genen, die unter beiden Differenzierung-Bedingungen, die die vorgeschlagenen in Richtung unabh&auml;ngiger Zelle Schicksale Einseitigkeit bereichert.

StemCellDB: Die menschliche Pluripotent Stem Cell Datenbank an den National Institutes of Health.

Molto l&#39;entusiasmo suscitato dalla tecnologia delle cellule staminali pluripotenti indotte &egrave; interessato la possibilit&agrave; di malattia modellazione cos&igrave; come il potenziale per la terapia cellulare personalizzato. Tuttavia, per perseguire questo &egrave; importante comprendere lo stato &#39;normale&#39; pluripotenti compresa sua variabilit&agrave; intrinseca. Abbiamo eseguito vari saggi di analisi molecolare per 21 linee di hESC e 8 hiPSC per generare uno snapshot completo dello stato indifferenziato di cellule staminali pluripotenti. Analisi dei dati di espressione genica ha rivelato nessun modello di espressione iPSC specifico gene conformemente a precedenti relazioni. Abbiamo confrontato ulteriormente le cellule, differenziate come corpi embryoid in 2 media proposto di avviare la differenziazione verso destini cella separata, come pure i tessuti adulti 20. Da questa analisi abbiamo abbiamo generato un elenco di gene che definisce la pluripotenza e stabilisce una linea di base per lo stato pluripotenti. Infine, forniamo liste di geni arricchiti sotto entrambe le condizioni di differenziazione che mostrano il bias proposto verso destini cellula indipendente.

StemCellDB: L&#39;umano cellule staminali pluripotenti Database presso i National Institutes of Health.

&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x6280;&#x8853;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x8208;&#x596E;&#x306E;&#x591A;&#x304F;&#x306F;&#x30D1;&#x30FC;&#x30BD;&#x30CA;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x7642;&#x6CD5;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x75BE;&#x60A3;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x61F8;&#x5FF5;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x308C;&#x3092;&#x8FFD;&#x6C42;&#x3059;&#x308B;&#x305D;&#x306E;&#x56FA;&#x6709;&#x306E;&#x5909;&#x52D5;&#x3092;&#x542B;&#x3080; &#39;normal&#39; &#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x69D8;&#x3005; &#x306A;&#x5206;&#x5B50;&#x30A2;&#x30C3;&#x30BB;&#x30A4; 21 hESC &#x7DDA;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x672A;&#x5206;&#x5316;&#x72B6;&#x614B;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x30B9;&#x30CA;&#x30C3;&#x30D7;&#x30B7;&#x30E7;&#x30C3;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B; 8 &#x304B;&#x3089;&#x4F5C;&#x88FD;&#x3057;&#x305F;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x89E3;&#x6790;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x306B;&#x5F93;&#x3063;&#x3066; iPSC &#x7279;&#x7570;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x8868;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3002;&#x6211;&#x3005; &#x306F;&#x3055;&#x3089;&#x306B;&#x500B;&#x5225;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x3068;&#x3057;&#x3066; 20 &#x6210;&#x4F53;&#x7D44;&#x7E54;&#x3078;&#x306E;&#x5206;&#x5316;&#x3092;&#x958B;&#x59CB;&#x3059;&#x308B;&#x63D0;&#x6848; 2 &#x306E;&#x30E1;&#x30C7;&#x30A3;&#x30A2;&#x306E;&#x80DA;&#x69D8;&#x4F53;&#x3068;&#x3057;&#x3066;&#x5206;&#x5316;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x3092;&#x6BD4;&#x8F03;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5206;&#x6790;&#x304B;&#x3089;&#x306F;&#x591A;&#x80FD;&#x6027;&#x3092;&#x5B9A;&#x7FA9;&#x3057;&#x3001;&#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x306E;&#x305F;&#x3081;&#x306E;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x78BA;&#x7ACB;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x63D0;&#x6848;&#x3055;&#x308C;&#x305F;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x3092;&#x793A;&#x3059;&#x4E21;&#x65B9;&#x306E;&#x5206;&#x5316;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x6FC3;&#x7E2E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

StemCellDB&#xFF1A; &#x56FD;&#x7ACB;&#x885B;&#x751F;&#x7814;&#x7A76;&#x6240;&#x3067;&#x306E;&#x3072;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x30C7;&#x30FC;&#x30BF;&#x30D9;&#x30FC;&#x30B9;&#x3002;

&#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xAE30;&#xC220;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC131; &#xB41C; &#xD765;&#xBD84;&#xC758; &#xC5F4;&#xAE30;&#xAC00; &#xB9CE;&#xC740; &#xC9C8;&#xBCD1; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C;, &#xB9DE;&#xCDA4;&#xD615;&#xB41C; &#xC138;&#xD3EC; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xB2A5;&#xC131; &#xBAA8;&#xB378;&#xB9C1;&#xC758; &#xAC00;&#xB2A5;&#xC131;&#xACFC; &#xD568;&#xAED8; &#xC6B0;&#xB824; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xAC83;&#xC744; &#xCD94;&#xAD6C; &#xD558;&#xB294; &#xADF8;&#xAC83;&#xC740; &#xADF8;&#xAC83;&#xC758; &#xB0B4;&#xC7AC; &#xBCC0;&#xB3D9;&#xC131;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#39;&#xC815;&#xC0C1;&#39; pluripotent &#xC0C1;&#xD0DC;&#xB97C; &#xC774;&#xD574; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; 21 hESC &#xB77C;&#xC778;&#xACFC; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; undifferentiated &#xC0C1;&#xD0DC;&#xC758; &#xC885;&#xD569;&#xC801;&#xC778; &#xC2A4;&#xB0C5;&#xC20F;&#xC744; &#xC0DD;&#xC131; 8 hiPSC &#xB77C;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBD84;&#xC790; &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xC218;&#xD589; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604; &#xB370;&#xC774;&#xD130; &#xBD84;&#xC11D; iPSC &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790; &#xC2DD; &#xD328;&#xD134;&#xC744; &#xB530;&#xB77C; &#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C;&#xB97C; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xC140;, 2 &#xBBF8;&#xB514;&#xC5B4; 20 &#xC131;&#xC778; &#xC870;&#xC9C1;&#xC73C;&#xB85C; &#xBCC4;&#xB3C4; &#xC140; &#xC6B4;&#xBA85;&#xC744; &#xD5A5;&#xD574; &#xCC28;&#xBCC4;&#xD654; &#xC2DC;&#xC791; &#xC81C;&#xC548; embryoid &#xC2DC;&#xCCB4;&#xB85C; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xBE44;&#xAD50;. &#xC774; &#xBD84;&#xC11D;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; &#xC815;&#xC758; pluripotency pluripotent &#xC0C1;&#xD0DC;&#xC5D0; &#xB300; &#xD55C; &#xCD08;&#xAE30; &#xACC4;&#xD68D;&#xC744; &#xC124;&#xC815; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBAA9;&#xB85D;&#xC744; &#xC0DD;&#xC131;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xB294; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; &#xD5A5;&#xD574; &#xC81C;&#xC548; &#xB41C; &#xBC14;&#xC774;&#xC5B4;&#xC2A4;&#xB97C; &#xBCF4;&#xC5EC; &#xB450; &#xBD84;&#xD654; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xB18D;&#xCD95; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790;&#xC758; &#xBAA9;&#xB85D;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

StemCellDB: &#xC778;&#xAC04;&#xC758; Pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB370;&#xC774;&#xD130;&#xBCA0;&#xC774;&#xC2A4; &#xAC74;&#xAC15;&#xC758; &#xAD6D;&#xB9BD; &#xC5F0;&#xAD6C;&#xC18C;&#xC5D0;&#xC11C;.

Gran parte de la emoci&oacute;n generada por la tecnolog&iacute;a de c&eacute;lulas madre pluripotentes inducidas se refiere a la posibilidad de enfermedad modelado as&iacute; como el potencial de la terapia celular personalizado. Sin embargo, para dedicarse a esto es importante comprender el estado pluripotente &#39;normal&#39;, incluyendo su variabilidad inherente. Hemos realizado varios ensayos perfiles moleculares para 21 hESCs y 8 hiPSC l&iacute;neas al generar una instant&aacute;nea completa del estado indiferenciado de c&eacute;lulas madre pluripotentes. El an&aacute;lisis de los datos de expresi&oacute;n gen&eacute;tica no revel&oacute; ning&uacute;n patr&oacute;n de expresi&oacute;n de genes espec&iacute;ficos de iPSC seg&uacute;n informes anteriores. Comparamos m&aacute;s c&eacute;lulas diferenciadas como cuerpos embryoid en 2 medios propuestos iniciar la diferenciaci&oacute;n hacia destinos separados de la c&eacute;lula, as&iacute; como 20 tejidos adultos. De este an&aacute;lisis hemos generado una lista del gen que define la pluripotencia y establece un punto de partida para el estado pluripotente. Por &uacute;ltimo, proporcionamos listas de genes enriquecidos en ambas condiciones de diferenciaci&oacute;n que demuestran el sesgo propuesto hacia destinos de c&eacute;lulas independientes.

StemCellDB: El humano Pluripotent Stem Cell base en los institutos nacionales de salud.

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this, we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However, these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level.

Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin.

Human pluripotent stem cells (hPSCs) provide powerful resources for application in regenerative medicine and pharmaceutical development. In the past decade, various methods have been developed for large-scale hPSC culture that rely on combined use of multiple growth components, including media containing various growth factors, extracellular matrices, 3D environmental cues, and modes of multicellular association. In this Protocol Review, we dissect these growth components by comparing cell culture methods and identifying the benefits and pitfalls associated with each one. We further provide criteria, considerations, and suggestions to achieve optimal cell growth for hPSC expansion, differentiation, and use in future therapeutic applications.

Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

The ability to differentiate induced pluripotent stem cells (iPSCs) into committed skeletal progenitors could allow for an unlimited autologous supply of such cells for therapeutic uses; therefore, we attempted to create novel bone-forming cells from human iPSCs using lines from two distinct tissue sources and methods of differentiation that we previously devised for osteogenic differentiation of human embryonic stem cells, and as suggested by other publications. The resulting cells were assayed using in vitro methods, and the results were compared with those obtained from in vivo transplantation assays. Our results show that true bone was formed in vivo by derivatives of several iPSC lines, but that the successful cell lines and differentiation methodologies were not predicted by the results of the in vitro assays. In addition, bone was formed equally well from iPSCs originating from skin or bone marrow stromal cells (also known as bone marrow-derived mesenchymal stem cells), suggesting that the iPSCs did not retain a "memory" of their previous life. Furthermore, one of the iPSC-derived cell lines formed verifiable cartilage in vivo, which likewise was not predicted by in vitro assays.

Article	Year
Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 11826117	2002
Overexpression of the 3'-untranslated region of myelin proteolipid protein mRNA leads to reduced expression of endogenous proteolipid mRNA. Neurochemical research\| PubMed ID: 12512940	2002
The mood stabilizer valproic acid stimulates GABA neurogenesis from rat forebrain stem cells. Journal of neurochemistry\| PubMed ID: 15379904	2004
Toward xeno-free culture of human embryonic stem cells. The international journal of biochemistry & cell biology\| PubMed ID: 16469522	2006
Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature biotechnology\| PubMed ID: 17572666	2007
Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells. Stem cells (Dayton, Ohio)\| PubMed ID: 22887864	2012
Non-colony type monolayer culture of human embryonic stem cells. Stem cell research\| PubMed ID: 22910561	2012
StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health. Stem cell research\| PubMed ID: 23117585	2012
Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin. Stem cell research\| PubMed ID: 24374290	2014
Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell stem cell\| PubMed ID: 24388173	2014
Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation. Stem cell research\| PubMed ID: 24603366	2014
Directed differentiation of human induced pluripotent stem cells toward bone and cartilage: in vitro versus in vivo assays. Stem cells translational medicine\| PubMed ID: 24855277	2014

Barbara S. Mallon

Biography

JoVE Journal Publications

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