Bing Lim's Laboratory
Genome Institute of Singapore, Stem Cell & Developmental Biology Group;
Harvard Medical School and Beth Israel Deaconess Medical Center, Division of Hematology/Oncology
Our laboratory has related research interests concerning embryonic stem cells, lineage reprogramming, microRNAs, and hematopoiesis. We exploit au courant
technology platforms inherent to the Genome Institute of Singapore to complement our research efforts. Our principal investigator, Dr. Bing Lim
, has dual appointments as both a Senior Group Leader at the Genome Institute of Singapore as well as a Associate Professor at Harvard Medical School.
I. Embryonic Stem Cells and Pluripotentiality
Pluripotent cells, such as embryonic stem (ES) cells, are the foundation of mammalian development - they may generate any of the hundreds of diverse cell types within the mammalian body. ES cells have since been exploited as a wellspring that may continually generate any bodily cell type (e.g. a neuron or a cardiomyocyte) that we might require for clinical, translational, or research purposes.
We are interested in two issues pertaining to the translational exploitation of ES cells: (1) What underlies the faculty of ES cells to differentiate into all fetal cell types? and (2) How do we direct the differentiation of ES cells into specific differentiated cell types of therapeutic relevance?
Embryonic, hematopoietic, and neural stem cells are transcriptionally dissimilar
Do ES cells rely on mechanisms similar to those exploited by multipotential neural stem cells (NSCs) and hematopoietic stem cells (HSCs) to differentiate into many different cell types? We resolved this long-standing question by demonstrating that ES cells, HSCs, and NSCs are transcriptionally divergent, thus demonstrating that separate stem cell populations likely dependent on different transcriptional contrivances to maintain their multilineage differentiation potential (Fortunel et al., 2003; Science
Identifying Sall4 as a novel, bona fide pluripotency transcription factor
Next, we focused specifically on what maintains the multilineage differentiation potential of ES cells. We identified an transcription factor, Sall4, that is required for ES cell pluripotentiality (Zhang et al., 2006; Nature Cell Biology
). We found that Sall4 maintains ES cells in an undifferentiated, uncommitted state by prohibiting trophectodermal differentiation. Moreover, we also found that Sall4 probably enables ES cells to differentiate into endodermal lineages. In subsequent studies, we found that Sall4 is expressed in both pluripotential ES cells as well as extraembryonic endoderm (XEN) cells and that unexpectedly, it is important for both pluripotential as well as extraembryonic cells (Lim et al., 2008; Cell Stem Cell
). We confirmed that Sall4 has divergent functions in ES cells versus XEN cells by mapping its genome-wide binding sites in both cell types using chromatin immunoprecipitation coupled to microarrays (chIP-chip).
A basis for the unlimited multilineage differentiation potential of ES cells
Studies such as our own have identified transcription factors (e.g., Sall4
) whose expression is required to maintain pluripotency; when bereft of these so-called "pluripotency factors", ES cells differentiate to specific lineages. These findings have led to the prevailing paradigm that pluripotency factors practice lineage-specific blockades on ES cell differentiation, and expression of a panoply of diverse pluripotency factors precludes ES cell differentiation to any downstream lineage, hence enabling undifferentiated stem cell self-renewal. Nevertheless, such a theoretical model does not provide an explanation for the most quintessenial property of an ES cell - its remarkable ability to liberally differentiate into any cell type within the mammalian body! To this end, we have recently averred that many classical pluripotency transcription factors expressed by ES cells (e.g., Oct4, Sox2, Nanog et al.
) indeed serve as lineage specification factors that provide ES cells with the ability to differentiate to specific fetal lineages (Loh and Lim, 2011; Cell Stem Cell
). Expression of these lineage specifiers within ES cells grants them with a representation of all the transcription factors necessary to prosecute differentiation into the major fetal lineages (definitive endoderm, mesoderm, and definitive ectoderm). Our proposal is substantiated by emergent studies that pluripotency factors genuinely appear to masquerade as bona fide
lineage specifiers; for example, Nanog specifies the differentiation of human ES cells to definitive endoderm while precluding specification to either mesoderm or neuroectoderm. Moreover, we predict that pluripotency is highly unstable due to continual conflict between pluripotency factors that are seeking to direct ES cell differentiation to specific lineages.
MicroRNAs specifying ES cell lineage commitment
Having defined components of the transcriptional regime that maintains the multilineage differentiation potential of ES cells, next we attempted to try to identify what mechanisms underlie ES cell differentiation and lineage specification. We identified three novel microRNAs that target the Oct4, Sox2, and Nanog coding sequences and are upregulated upon ES cell differentiation (Tay et al., 2008; Nature
). Thus, it appears that ES cell lineage specification relies on the upregulation of microRNAs to suppress ES cell transcription factors that would otherwise inhibit differentiation. We also found another microRNA, miR-134, that directly specifies neuroectodermal commitment of ES cells via inhibition of Nanog and Nr5a2 protein translation (Tay et al., 2008; Stem Cells
Tcf3 is required to prosecute ES cell differentiation
Furthermore, we also found an unexpected role for the transcription factor Tcf3 (the terminal effector of Wnt/β-catenin signaling) in conferring ES cells with the ability to differentiate, as knockdown of Tcf3 prevents ES cells from differentiating (Tam et al., 2008; Stem Cells
). Such results were unanticipated, given previous high profile reports that Wnt/β-catenin signaling directs the undifferentiated self-renewal of ES cells. We demonstrated that Tcf3 likely orchestrates differentiation by downregulating Oct4 and Nanog while concomitantly upregulating a plethora of genes associated with lineage commitment.
Enabling undifferentiated self-renewal of human ES cells
Moving from mouse ES cells to therapeutically relevant human ES cells, we used microarray analyses to identify growth factor receptors expressed on the surface of human ES cells that might be important for regulating their self-renewal (Soh et al., 2008; Stem Cells
). From these analyses, we identified that the pleiotrophin receptor is expressed on human ES cells that it is necessary for their self-renewal. From a practical perspective, addition of exogenous pleiotrophin enhances human ES cell propagation, making it useful for long-term human ES cell culture.
II. Lineage Reprogramming
Parsimoniously speaking, what makes one cell type different from another is lineage-specific transcription factor regimes that impart unique lineages with their unique gene expression programs. Such a paradigm would suggest that a cell could be directly reprogrammed to an alternative lineage by simply substituting its current cadre of transcription factors and epigenetic regulators with those that are expressed by another cell type. Indeed, this appears to be the case, as fibroblasts may be directly reprogrammed into induced pluripotent stem (iPS) cells, cardiomyocytes, neurons, or myocytes by simple overexpression of transcription factors that are expressed in these target cell types. Given our experience with pluripotential cells, an ongoing research focus in our lab is to reprogram differentiated cells into pluripotential iPS cells.
Efficient generation of "high quality" mouse iPS cells with Tbx3
Despite the recent excitement over iPS cells, a word of caution comes from the fact that nearly the majority of present (mouse) iPS cell lines are incapable of tetraploid complementation and thus are not bona fide pluripotential cells (Loh and Lim, 2010; Cell Stem Cell
). With such thinking in mind, we set out to try to devise a method by which we could homogenously generate “high quality” iPS cells that are authentically pluripotent and are capable of tetraploid and diploid complementation. We found that overexpression of transcription factor Tbx3 reproducibly enables the creation of high quality mouse iPS cells with an enhanced capacity for diploid complementation, germline transmission, as well as tetraploid complementation (Han et al., 2010; Nature
). This was the first such demonstration that the “quality” of iPS cells could be deterministically enhanced by defined factors.
Our present understanding of cell fate and physiology, especially in regards to pluripotential stem cells, is heavily focused on transcription factors. In our laboratory, we have sought to explore further the role of microRNAs in specific cell types of interest. As aforementioned, we have defined novel roles for various microRNAs in ES cell differentiation (Tay et al., 2008; Nature
and Tay et al., 2008; Stem Cells
). We also developed a novel algorithm to find the targets of microRNAs, and through this, we found that many microRNAs may have thousands of target transcripts, far more than previously believed (Miranda et al., 2006; Cell
We also identified the first microRNA known to regulate the tumor suppressor p53—miR-125b (Le et al., 2009; Genes & Development
). miR-125b downregulation is essential for activation of p53-orchestrated stress responses, and we also found an unanticipated role for it in regulating neuronal differentiation, as it is specifically expressed in the brain (Le et al., 2009; Molecular and Cellular Biology
Our principal investigator—Dr. Bing Lim—did his graduate training with Earnest McCulloch, co-discoverer of hematopoietic stem cells. Thus, we have a consequential interest in hematopoietic stem cells and various other aspects of the hematopoietic system. Through a screen for genes preferentially expressed in hematopoietic cells, we identified and cloned D4 GDI (GDID4/RhoGDIβ), a member of the family of Rho GTPase guanosine disassociation inhibitors (Lelias et al., 1993; Proc Natl Acad Sci
). Gdid4 deletion does not impair hematopoietic differentiation, but rather, Gdid4—/— macrophages are impaired in their superoxide production (Guillemot et al., 1996; Blood
). Later, we discovered a closely related homolog to GDID4, which we named RhoGDIγ (Adra et al., 1997; Proc Natl Acad Sci
). During our investigations of Gdid4 function, we noticed that Gdid4—/— mice often developed an autoimmune disease similar to the human disease systemic lupus erythematosus (Layer et al., 2003; Immunity
). We were surprised to find that this autoimmune disorder was not the consequence of Gdid4 deficiency, but rather that these Gdid4—/— mice developed autoimmunity due to a spontaneous and unrelated mutation in the Ras guanine exchange factor Rasgrp1. This Rasgrp1 mutation was found to block Ras signaling, and led to the activation of B cells and autoantibody production, thus providing an excellent murine model for the lupus disorder.
We have also recently found that downregulation of c-Myc is required for the terminal differentiation of erythroblasts during erythropoiesis (Jayapal et al., 2010; J. Biol Chem
). c-Myc inhibits nuclear condensation and enucleation of erythrocytes, and thus its downregulation is required for proper terminal differentiation.
We take a collaborative approach in all our research enterprises, and many of the above research achievements would have been impossible if not for the collaboration and support tendered by various investigators at Singapore, the United States, and elsewhere. In addition, we have been pleased to help many other research groups in their own research efforts.
Our list of recent collaborators includes but is certainly not limited to—Dr. Harvey Lodish
(MIT/Whitehead: Le et al., 2009; Genes & Development; Le et al., 2009; Molecular Cellular Biology), Dr. Ludovic Vallier
(Cambridge: Soh et al., 2008; Stem Cells), Dr. Henry Yang
(A*STAR: Lim et al., 2008; Cell Stem Cell; Han et al., 2010; Nature), Dr. Huck-Hui Ng
(A*STAR: Chia et al., 2010; Nature; Heng et al., 2010; Cell Stem Cell; Feng et al., 2009; Nature Cell Biology), and Dr. Isidore Rigoutsos
(IBM: Tay et al., 2008; Nature; Tay et al., 2008; Stem Cells).
- Zhang L, Flygare J, Wong P, Lim B*, Lodish HF* "miR-191 regulates mouse erythroblast enucleation by down-regulating Riok3 and Mxi1." Genes Dev 2011 Jan 15 ; 25(2) : 119-24 Epub 2010 Dec 31 Abstract
- Loh KM and Lim B* "A precarious balance: pluripotency factors as lineage specifiers." Cell Stem Cell 2011 Apr 8 ; 8(4) : 363-9 Abstract
- Han J, Yuan P, Yang H, Zhang J, Soh BS, Li P, Lim SL, Cao S, Tay J, Orlov YL, Lufkin T, Ng HH, Tam WL, Lim B* "Tbx3 improves the germ-line competency of induced pluripotent stem cells." Nature 2010 Feb 25 ; 463(7284) : 1096-100 Epub 2010 Feb 7 Abstract
- Loh KM and Lim B* "Recreating pluripotency?" Cell Stem Cell 2010 Aug 6 ; 7(2) : 137-9 Abstract
- Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V, Lodish HF*, Lim B* "MicroRNA-125b is a novel negative regulator of p53." Genes Dev 2009 Apr 1 ; 23(7) : 862-76 Epub 2009 Mar 17 Abstract
- Lim CY, Tam WL, Zhang J, Ang HS, Jia H, Lipovich L, Ng HH, Wei CL, Sung WK, Robson P, Yang H, Lim B* "Sall4 regulates distinct transcription circuitries in different blastocyst-derived stem cell lineages." Cell Stem Cell 2008 Nov 6 ; 3(5) : 543-54 Epub 2008 Sep 18 Abstract
- Tay Y, Zhang J, Thomson AM*, Lim B*, Rigoutsos I* "MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation." Nature 2008 Oct 23 ; 455(7216) : 1124-8 Epub 2008 Sep 17 Abstract
- Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B*, Rigoutsos I* "A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes." Cell 2006 Sep 22 ; 126(6) : 1203-17 Abstract
- Zhang J, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, Lou Y, Yang J, Ma Y, Chai L, Ng HH, Lufkin T, Robson P, Lim B* "Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1." Nat Cell Biol 2006 Oct ; 8(10) : 1114-23 Epub 2006 Sep 17 Abstract
- Fortunel NO, Otu HH, Ng HH, Chen J, Mu X, Chevassut T, Li X, Joseph M, Bailey C, Hatzfeld JA, Hatzfeld A, Usta F, Vega VB, Long PM, Libermann TA, Lim B* "Comment on "Stemness: transcriptional profiling of embryonic and adult stem cells" and "A stem cell molecular signature"." Science 2003 Oct 17 ; 302(5644) : 393; author reply 393 Abstract