Wenlin Li

Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.

(Cell Stem Cell 4, 16–19; January 9, 2009)In our recent article, we unfortunately misquoted the findings in a recent study by Ying et al. (2008)xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al. (2008). When describing previous work using combination of the MEK inhibitor PD0325901 and the GSK3b inhibitor CHIR99021, our statement “Recent studies demonstrated that addition of the FGFR inhibitor PD173074 to the above cocktail is sufficient to maintain mESC pluripotency in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008).” was not accurate. Ying et al., in fact, used PD0325901 to replace PD1730474 and demonstrated maintenance of mESCs in the absence of LIF with these two factors only.The corrected section reads “Indeed, after serial passages, the growth of putative riPSCs treated with only PD0325901 and CHIR99021 declined, and the culture deteriorated due to the expansion of differentiated cells, although recent studies demonstrated that this combination of inhibitors can be used to maintain mESC self-renewal in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008). Because the TGFβ/Activin A/Nodal signaling cascade is essential to maintain undifferentiated hESCs and EpiSCs, but dispensable for mESC self-renewal, we tested whether the addition of an inhibitor of the type 1 TGFβ receptor, ALK5 (A-83-01), could help stabilize our riPSC cultures.”In addition, we omitted to mention that while our study was under review, Silva et al. (2008)xPromotion of Reprogramming to Ground State Pluripotency by Signal Inhibition. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. PLoS Biol. 2008; 6: 2237–2247Crossref | Scopus (456)See all ReferencesSilva et al. (2008) also published the use of these two inhibitors in the generation and propagation of mouse iPSCs.We apologize for any confusion caused.

A chemical platform for improved induction of human iPSCs

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

Reprogramming of Human Primary Somatic Cells by OCT4 and Chemical Compounds

Induced pluripotent stem cell (iPSC) technology, i.e. reprogramming somatic cells into pluripotent cells that closely resemble embryonic stem cells (ESCs) by introduction of defined transcription factors (TFs), holds great potential in biomedical research and regenerative medicine (Takahashi et al., 2006; Takahashi et al., 2007; Yu et al., 2007). Various strategies have been developed to generate iPSCs with fewer or no exogenous genetic manipulations, which represent a major hurdle for iPSC applications (Yamanaka et al., 2009). With the ultimate goal of generating iPSCs with a defined small molecule cocktail alone, substantial effort and progress have been made in identifying chemical compounds that can functionally replace exogenous reprogramming TFs and/or enhance the efficiency and kinetics of reprogramming (Shi et al., 2008; Huangfu et al., 2008; Lyssiotis et al., 2009; Ichida et al., 2009; Maherali et al., 2009; Lin et al., 2009; Li et al., 2009; Esteban et al., 2010). To date, only neural stem cells (NSCs), which endogenously express SOX2 and cMYC at a high level, have been reprogrammed to iPSCs by exogenous expression of just OCT4 (Kim et al., 2009). However, human fetal NSCs are rare and difficult to obtain. It is therefore important to develop reprogramming conditions for other more accessible somatic cells. Here we report a small molecule cocktail that enables reprogramming of human primary somatic cells to iPSCs with exogenous expression of only OCT4. In addition, mechanistic studies revealed that modulation of cell metabolism from mitochondrial oxidation to glycolysis plays an important role in reprogramming.

Generation of Human Induced Pluripotent Stem Cells in the Absence of Exogenous Sox2

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here, we report that a specific glycogen synthase kinase 3 (GSK‐3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine‐specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4. To our knowledge, this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK‐3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming. STEM CELLS 2009;27:2992–3000

Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.

Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFβ pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

Chemical approaches to stem cell biology and therapeutics

Small molecules that modulate stem cell fate and function offer significant opportunities that will allow the full realization of the therapeutic potential of stem cells. Rational design and screening for small molecules have identified useful compounds to probe fundamental mechanisms of stem cell self-renewal, differentiation, and reprogramming and have facilitated the development of cell-based therapies and therapeutic drugs targeting endogenous stem and progenitor cells for repair and regeneration. Here, we will discuss recent scientific and therapeutic progress, as well as new perspectives and future challenges for using chemical approaches in stem cell biology and regenerative medicine.

Small molecules, big roles – the chemical manipulation of stem cell fate and somatic cell reprogramming

Summary Despite the great potential of stem cells for basic research and clinical applications, obstacles – such as their scarce availability and difficulty in controlling their fate – need to be addressed to fully realize their potential. Recent achievements of cellular reprogramming have enabled the generation of induced pluripotent stem cells (iPSCs) or other lineage-committed cells from more accessible and abundant somatic cell types by defined genetic factors. However, serious concerns remain about the efficiency and safety of current genetic approaches to cell reprogramming and traditional culture systems that are used for stem cell maintenance. As a complementary approach, small molecules that target specific signaling pathways, epigenetic processes and other cellular processes offer powerful tools for manipulating cell fate to a desired outcome. A growing number of small molecules have been identified to maintain the self-renewal potential of stem cells, to induce lineage differentiation and to facilitate reprogramming by increasing the efficiency of reprogramming or by replacing genetic reprogramming factors. Furthermore, mechanistic investigations of the effects of these chemicals also provide new biological insights. Here, we examine recent achievements in the maintenance of stem cells, including pluripotent and lineage-specific stem cells, and in the control of cell fate conversions, including iPSC reprogramming, conversion of primed to naïve pluripotency, and transdifferentiation, with an emphasis on manipulation with small molecules.

Concise Review: A Chemical Approach to Control Cell Fate and Function†‡§

Stem cells are essential for maintaining tissue homeostasis and mediating physiological and pathological regeneration. Recent breakthroughs in stem cell biology have generated tremendous enthusiasm and hope for the therapeutic potential of stem cells in regenerative medicine. However, this research is still in an early development stage. An improved understanding of stem cell biology is required to precisely manipulate stem cell fate and to harness these cells for regenerative medicine. Small molecules, targeting specific signaling pathways and mechanisms, are powerful tools for manipulating stem cells for desired outcomes. Those small molecules are increasingly important in probing the fundamental mechanisms of stem cell biology and facilitating the development of therapeutic approaches for regenerative medicine. These could involve cell replacement therapies with homogenous functional cells produced under chemically defined conditions in vitro and the development of small‐molecule drugs that modulate patients endogenous cells for therapeutic benefit. STEM CELLS 2012;30:61–68

Diethylation labeling combined with UPLC/MS/MS for simultaneous determination of a panel of monoamine neurotransmitters in rat prefrontal cortex microdialysates.

The primary challenge associated with the development of an LC/MS/MS-based assay for simultaneous determination of biogenic monoamine neurotransmitters such as norepinephrine (NE), dopamine (DA), serotonin (5-HT), and normetanephrine (NM) in rat brain microdialysates is to improve detection sensitivity. In this work, a UPLC/ MS/MS-based method combined with a diethyl labeling technique was developed for simultaneous determination of a panel of monoamines in rat prefrontal cortex microdialysates. The chromatographic run time is 3.5 min/ sample. The limits of detection of the UPLC/MS/MS-based method for NE, DA, 5-HT/ and NM, with/without diethyl labeling of monoamines, are 0.005/0.4 (30/2367 pM), 0.005/0.1 (33/653 pM), 0.005/0.2 (28/1136 pM), and 0.002/0.2 ng/mL (11/1092 pM), respectively. Diethyl labeling of amino groups of monoamines affords 20-100 times increased detection sensitivity of corresponding native monoamines during the UPLC/MS/MS analysis. This could result from the following: (1) improved fragmentation patterns; (2) increased hydrophobicity and concomitantly increased ionization efficiency in ESI MS and MS/MS analysis; (3) reduced matrix interference. This labeling reaction employs a commercially available reagent, acetaldehyde-d4, to label the amine groups on the monoamines via reductive amination. It is also simple, fast (approximately 25-min reaction time), specific, and quantitative under mild reaction conditions. Data are also presented from the application of this assay to monitor the drug-induced changes of monoamine concentrations in rat prefrontal cortex microdialysate samples followed by administration of SKF 81297, a selective D1 dopamine receptor agonist known to elevate the extracellular level of the neurotransmitters DA and NE in the central nervous system.