generation-induced-pluripotent-stem-cells-from-frozen-buffy-coats

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Sanford-Burnham Medical Research Institute

Unlike other N-methyl-D-aspartate receptor (NMDAR) antagonists, clinical trials have shown that memantine is clinically tolerated and effective in the treatment of Alzheimer's disease. The mechanism for memantine tolerability, however, remains contentious but may be partly explained by its uncompetitive antagonism. The specific site of memantine block in the NMDAR channel interacts with magnesium and is assumed to be at or near a narrow constriction representing the channel selectivity filter. A second, very low-affinity site of memantine action has also been reported. Here, using mutational analysis and substituted cysteine accessibility methods on recombinant NR1/NR2A NMDARs expressed in Xenopus oocytes, we precisely localize both the specific and second memantine-blocking sites. Intriguingly, memantine interacts with its specific blocking site in the same fashion as intracellular rather than extracellular Mg(2+). Thus, the N-site asparagine (N) in the M2 region of the NR1 subunit represents the dominant site for uncompetitive antagonism by memantine. The N and N + 1 site asparagines in NR2A produce strong electrostatic interactions with memantine. In contrast, the second (superficial) memantine-blocking site, located at the extracellular vestibule of the channel, appears to be nonspecific and overlaps the site occupied by the nonspecific pore blocker hexamethonium. Residues in the post-M3 segment of the NR1 subunit are not directly involved in memantine binding. The distinct patterns of interaction and the relative degree of affinity of memantine for these two binding sites contribute to the drug's excellent pharmacological profile of clinical tolerability. In the future, these parameters should be considered in searching for improved neuroprotective agents in this class.

Pharmacological implications of two distinct mechanisms of interaction of memantine with N-methyl-D-aspartate-gated channels.

Spontaneous migration of the site of 2:1 atrioventricular block during atrioventricular nodal reentrant tachycardia.

Most neuroprotective drugs have failed in clinical trials because of side-effects, causing normal brain function to become compromised. A case in point concerns antagonists of the N-methyl-D-aspartate type of glutamate receptor (NMDAR). Glutamate receptors are essential to the normal function of the central nervous system. However, their excessive activation by excitatory amino acids, such as glutamate itself, is thought to contribute to neuronal damage in many neurological disorders ranging from acute hypoxic-ischemic brain injury to chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. The dual role of NMDARs in particular for normal and abnormal functioning of the nervous system imposes important constraints on possible therapeutic strategies aimed at ameliorating neurological diseases. Blockade of excessive NMDAR activity must therefore be achieved without interference with its normal function. In general, NMDAR antagonists can be categorized pharmacologically according to the site of action on the receptor-channel complex. These include drugs acting at the agonist (NMDA) or co-agonist (glycine) sites, channel pore, and modulatory sites, such as the S-nitrosylation site where nitric oxide (NO) reacts with critical cysteine thiol groups. Because glutamate is thought to be the major excitatory transmitter in the brain, generalized inhibition of a glutamate receptor subtype like the NMDAR causes side-effects that clearly limit the potential for clinical applications. Both competitive NMDA and glycine antagonists, even although effective in preventing glutamate-mediated neurotoxicity, will cause generalized inhibition of NMDAR activities and thus have failed in many clinical trials. Open-channel block with the property of uncompetitive antagonism is the most appealing strategy for therapeutic intervention during excessive NMDAR activation as this action of blockade requires prior activation of the receptor. This property, in theory, leads to a higher degree of channel blockade in the presence of excessive levels of glutamate and little blockade at relatively lower levels, for example, during physiological neurotransmission. Utilizing this molecular strategy of action, we review here the logical process that we applied over the past decade to help develop memantine as the first clinically tolerated yet effective agent against NMDAR-mediated neurotoxicity. Phase 3 (final) clinical trials have shown that memantine is effective in treating moderate-to-severe Alzheimer's disease while being well tolerated. Memantine is also currently in trials for additional neurological disorders, including other forms of dementia, glaucoma, and severe neuropathic pain. Additionally, taking advantage of memantine's preferential binding to open channels and the fact that excessive NMDAR activity can be down-regulated by S-nitrosylation, we have recently developed combinatorial drugs called NitroMemantines. These drugs use memantine as a homing signal to target NO to hyperactivated NMDARs in order to avoid systemic side-effects of NO such as hypotension (low blood pressure). These second-generation memantine derivatives are designed as pathologically activated therapeutics, and in preliminary studies appear to have even greater neuroprotective properties than memantine.

The chemical biology of clinically tolerated NMDA receptor antagonists.

Under ambient air conditions, NO inhibits NMDAR activity by reacting with the NR2A subunit C399 along with two additional cysteine pairs if their disulfide bonds are reduced to free thiol groups [NR1(C744,C798); NR2(C87,C320)]. Here we demonstrate that relative hypoxia enhances S-nitrosylation of NMDARs by a unique mechanism involving an "NO-reactive oxygen sensor motif" whose determinants include C744 and C798 of the NR1 subunit. Redox reactions involving these two thiol groups sensitize other NMDAR sites to S-nitrosylation and consequent receptor inhibition, while their own nitrosylation has little effect on NMDAR activity. The crystal structure of the ligand-binding domain of NR1 reveals a flexible disulfide bond (C744-C798), which may account for its susceptibility to reduction and subsequent reaction with NO that is observed with biochemical techniques. These thiols may be nitrosylated preferentially during increasing hypoxia or stroke conditions, thus preventing excessive activity associated with cytotoxicity while avoiding blockade of physiologically active NMDARs.

Hypoxia enhances S-nitrosylation-mediated NMDA receptor inhibition via a thiol oxygen sensor motif.

Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.

Electrophysiological challenges of cell-based myocardial repair.

Glutamate is the major excitatory neurotransmitter in the brain. The NMDA subtype of glutamate receptors (NMDAR) is known to mediate many physiological neural functions. However, excessive activation of NMDARs contributes to neuronal damage in various acute and chronic neurological disorders. To avoid unwanted adverse side effects, blockade of excessive NMDAR activity must therefore be achieved without affecting its physiological function. Memantine, an adamantane derivative, has been used for the treatment of Alzheimer's disease with an excellent clinical safety profile. We previously showed that memantine preferentially blocked neurotoxicity mediated by excessive NMDAR activity while relatively sparing normal neurotransmission, in part because of its uncompetitive antagonism with a fast off-rate. Here, using rat autaptic hippocampal microcultures, we show that memantine at therapeutic concentrations (1-10 microM) preferentially blocks extrasynaptic rather than synaptic currents mediated by NMDARs in the same neuron. We found that memantine blocks extrasynaptic NMDAR-mediated currents induced by bath application of 100 microM NMDA/10 microM glycine with a twofold higher potency than its blockade of the NMDAR component of evoked EPSCs (EPSCs(NMDAR)); this effect persists under conditions of pathological depolarization in the presence of 1 mm extracellular Mg(2+). Thus, our findings provide the first unequivocal evidence to explain the tolerability of memantine based on differential extrasynaptic/synaptic receptor blockade. At therapeutic concentrations, memantine effectively blocks excessive extrasynaptic NMDAR-mediated currents, while relatively sparing normal synaptic activity.

Memantine preferentially blocks extrasynaptic over synaptic NMDA receptor currents in hippocampal autapses.

Cardiac progenitor cells (CPCs) are multipotent cells that may offer tremendous potentials for the regeneration of injured myocardium. To expand the limited number of CPCs for effective clinical regeneration of myocardium, it is important to understand their proliferative potentials. Single-cell based assays were utilized to purify c-kit(pos) CPCs from human and mouse hearts. MicroRNA profiling identified eight differentially expressed microRNAs in CPCs from neonatal and adult hearts. Notably, the predicted protein targets were predominantly involved in cellular proliferation-related pathways. To directly test this phenotypic prediction, the developmental variance in the proliferation of CPCs was tested. Ki67 protein expression and DNA kinetics were tested in human and mouse in vivo CPCs, and doubling times were tested in primary culture of mouse CPCs. The human embryonic and mouse neonatal CPCs showed a six-fold increase in Ki67 expressing cells, a two-fold increase in the number of cells in S/G2-M phases of cell cycle, and a seven-fold increase in the doubling time in culture when compared to the corresponding adult CPCs. The over-expression of miR-17-92 increased the proliferation in adult CPCs in vivo by two-fold. In addition, the level of retinoblastoma-like 2 (Rbl2/p130) protein was two-fold higher in adult compared to neonatal-mouse CPCs. In conclusion, we demonstrate a differentially regulated cohort of microRNAs that predicts differences in cellular proliferation in CPCs during postnatal development and target microRNAs that are involved in this transition. Our study provides new insights that may enhance the utilization of adult CPCs for regenerative therapy of the injured myocardium.

MicroRNA profiling predicts a variance in the proliferative potential of cardiac progenitor cells derived from neonatal and adult murine hearts.

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26â€‰years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased Î²-catenin activity in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-Î³) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-Î³ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2â€‰months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.

Cellulaire de reprogrammation de cellules somatiques &agrave; cellules souches pluripotentes induites sp&eacute;cifiques au patient (CISP) permet de mod&eacute;lisation in vitro des humains troubles g&eacute;n&eacute;tiques par enqu&ecirc;tes pathog&egrave;nes et &eacute;crans th&eacute;rapeutiques. Toutefois, &agrave; l&#39;aide de cardiomyocytes d&eacute;riv&eacute;s iPSC (iPSC-CMs) pour mod&eacute;liser une cardiopathie adulte reste difficile en raison de l&#39;incertitude quant &agrave; la capacit&eacute; du CISP-CMs relativement immatures &agrave; r&eacute;capituler compl&egrave;tement ph&eacute;notypes maladies adultes. Cardiomyopathie ventriculaire droite arythmog&egrave;ne dysplasie / (ARVD/C) est une maladie cardiaque h&eacute;r&eacute;ditaire caract&eacute;ris&eacute;e par une perte grasse pathologique de l&#39;infiltration et les myocytes cardiaques se situe principalement dans le ventricule droit, qui est associ&eacute; &agrave; des arythmies ventriculaires mortelles. Plus de 50 % des personnes touch&eacute;es ont desmosome mutations g&eacute;niques, plus souvent en PKP2, encodage plakophiline-2 (R&eacute;f. 9). L&#39;&acirc;ge m&eacute;dian &agrave; la pr&eacute;sentation d&#39;ARVD/C est de 26 ans. Nous avons utilis&eacute; des m&eacute;thodes publi&eacute;es ant&eacute;rieurement pour g&eacute;n&eacute;rer des lignes de l&#39;iPSC de fibroblastes de deux patients pr&eacute;sentant des mutations ARVD/C et PKP2. Mutants PKP2 iPSC-CMs d&eacute;montrent translocation nucl&eacute;aire plakoglobine anormale et une diminution de &beta;-cat&eacute;nine activit&eacute; cardiog&eacute;nique conditions ; Pourtant, ces caract&eacute;ristiques anormales ne suffisent pas &agrave; reproduire les ph&eacute;notypes pathologiques d&#39;ARVD/C dans des conditions normalis&eacute;es cardiog&eacute;nique. Ici, nous montrons que l&#39;induction de l&#39;adulte type m&eacute;tabolique &eacute;nerg&eacute;tique d&#39;un embryon/glycolytique &Eacute;tat et activation d&#39;anormale peroxisome proliferator-activated receptor gamma (PPAR-&gamma;) sous-tendent la pathog&eacute;nie d&#39;ARVD/C. Par co-activating le m&eacute;tabolisme normal des PPAR-alpha-d&eacute;pendante et voie anormale de PPAR-&gamma; en battant corps embryo&iuml;des (EBs) avec les milieux d&eacute;finis, nous avons &eacute;tabli un ARVD/C efficace in vitro mod&egrave;le dans les 2 mois. Ce mod&egrave;le manifeste la lipogen&egrave;se exag&eacute;r&eacute;e et l&#39;apoptose dans mutant PKP2 iPSC-iPSC-CMs CMs. avec une mutation homozygote de PKP2 a &eacute;galement eu des d&eacute;ficits de calcium-manutention. Notre &eacute;tude est la premi&egrave;re &agrave; d&eacute;montrer que l&#39;induction du m&eacute;tabolisme de type adulte a un r&ocirc;le essentiel dans l&#39;&eacute;tablissement d&#39;un mod&egrave;le de maladie d&#39;adulte-d&eacute;but &agrave; l&#39;aide de CISP sp&eacute;cifique au patient. En utilisant ce mod&egrave;le, nous avons r&eacute;v&eacute;l&eacute; cruciales insights pathog&egrave;nes que d&eacute;rangement m&eacute;tabolique en milieu m&eacute;tabolique de type adulte est &agrave; la base des pathologies ARVD/C, ce qui nous permet de proposer de nouvelles strat&eacute;gies th&eacute;rapeutiques modificateurs de la maladie.

&Eacute;tudier la dysplasie ventriculaire droite arythmog&egrave;ne avec CISP sp&eacute;cifique au patient.

Cellulare riprogrammazione di cellule somatiche a cellule staminali pluripotenti indotte paziente-specifici (iPSCs) permette di modellazione in vitro di malattie genetiche umane per indagini patogene e schermi terapeutici. Tuttavia, usando iPSC derivate cardiomyocytes (iPSC-CMs) per modellare una malattia di cuore di adulto-inizio rimane impegnativo a causa delle incertezze per quanto riguarda la capacit&agrave; di riassumere pienamente fenotipi adulti malattia relativamente immaturo iPSC-CMs. Cardiomiopatia displasia ventricolare destra aritmogena (ARVD/C) &egrave; una malattia di cuore ereditaria caratterizzata dalla perdita grassa infiltrazione e cardiomyocyte patologica prevalentemente nel ventricolo destro, che &egrave; associato con aritmie ventricolari letali. Oltre il 50% delle persone affette hanno desmosome mutazioni del gene, pi&ugrave; comunemente in PKP2, codifica placofilina-2 (Rif. 9). L&#39;et&agrave; mediana alla presentazione di ARVD/C &egrave; 26 anni. Abbiamo utilizzato metodi precedentemente pubblicati per generare iPSC linee dai fibroblasti dei due pazienti con mutazioni di ARVD/C e PKP2. Mutante PKP2 iPSC-CMs dimostrare la traslocazione nucleare plakoglobina anormale e attivit&agrave; di &beta;-catenina &egrave; diminuito in condizioni cardiogenica; Eppure, queste caratteristiche anomale sono insufficienti per riprodurre i fenotipi patologici di ARVD/C in condizioni standard cardiogeno. Qui vi mostriamo che induzione dell&#39;adulto-come energetica metabolica da un&#39;embrionale/glicolitico stato e attivazione di anormale peroxisome proliferator-activated receptor gamma (PPAR-&gamma;) sono alla base della patogenesi della ARVD/C. Di co-activating il normale metabolismo di PPAR-alfa-dipendente e anormale pathway PPAR-&gamma; nel battere embryoid corpi (EBs) con mezzi definiti, abbiamo stabilito un efficiente ARVD/C in vitro modello entro 2 mesi. Questo modello si manifesta esagerato lipogenesi e l&#39;apoptosi in mutante PKP2 iPSC-CMs. iPSC-CMs con una mutazione omozigote PKP2 aveva anche deficit di calcio-gestione. Il nostro studio &egrave; il primo a dimostrare che l&#39;induzione del metabolismo adulto-come ha un ruolo fondamentale nella creazione di un modello di malattia ad esordio adulto utilizzando iPSCs paziente-specifici. Utilizzando questo modello, abbiamo rivelato cruciale intuizioni patogeni che derangement metabolico nel milieu metabolico adulto-come sottolinea ARVD/C patologie, che ci permette di proporre nuove strategie terapeutiche modificanti la malattia.

Studiando la displasia ventricolare destra aritmogena con iPSCs paziente-specifici.

Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy.

Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.

Arrhythmogenic right ventricular dysplasia back in force.

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.

A long noncoding RNA protects the heart from pathological hypertrophy.

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in-vitro modeling of human cardiac disorders for pathogenic and therapeutic investigations. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging because of the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia (ARVD) is an inherited cardiomyopathy characterized by pathological fibrofatty infiltration and cardiomyocyte (CM) loss predominantly in the right ventricle (RV), leading to heart failure and lethal arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly inPKP2encoding plakophilin-2. Using Yamanaka's pluripotent factors, we generated iPSC lines from ARVD patients withPKP2mutations. We first developed a method to induce metabolic maturation of iPSC-CMs and showed that induction of adult-like metabolic energetics from an embryonic/glycolytic state is essential to model an adult-onset cardiac disease using patient-specific iPSCs. Furthermore, we showed that coactivation of normal peroxisome proliferator-activated receptor (PPAR)-α and abnormal PPARγ pathways in ARVD iPSC-CMs resulted in exaggerated CM lipogenesis, CM apoptosis, Na(+)channel downregulation and defective intracellular calcium handling, recapitulating the pathological signatures of ARVD. Using this model, we revealed novel pathogenic insights that metabolic derangement in an adult-like metabolic milieu underlies ARVD pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

Huei-Sheng Vincent Chen

Del E. Webb Center for Neuroscience,
Aging & Stem Cell Research,
Del E. Webb Center for Neuroscience, Aging & Stem Cell Research

Sanford-Burnham Medical Research Institute

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Huei-Sheng Vincent Chen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Del E. Webb Center for Neuroscience,Aging & Stem Cell Research,Del E. Webb Center for Neuroscience, Aging & Stem Cell Research

Sanford-Burnham Medical Research Institute

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Del E. Webb Center for Neuroscience,
Aging & Stem Cell Research,
Del E. Webb Center for Neuroscience, Aging & Stem Cell Research