Stem Cell Lineage Specification Research Articles

Hypothermia-Modulating Matrix Elasticity of Injured Brain Promoted Neural Lineage Specification of Mesenchymal Stem Cells

Both chemical and physical microenvironments appear to be important for lineage specification of umbilical cord mesenchymal stem cells (UCMSCs). However, physical factors such as the elastic modulus in traumatic brain injury (TBI) are seldom studied. Intracranial hypertension and cerebral edema after TBI may change the brain’s physical microenvironment, which inhibits neural lineage specification of transplanted UCMSCs. The purpose of this study is to investigate the potential regulatory effect of mild hypothermia on the elastic modulus of the injured brain. First, we found that more UCMSCs grown on gels mimicking the elastic modulus of the brain (0.5 kPa) differentiated into neural cells, which were verified with the formation of branched cells and the expression of neural markers. Then, UCMSCs were transplanted into TBI rats, and we observed that mild hypothermia resulted in the differentiation of more neurons and astrocytes from transplanted UCMSCs. To demonstrate that more neural specification of UCMSCs was due to the regulation of the elastic modulus, we monitored intracranial pressure and cerebral edema. The results showed that mild hypothermia significantly reduced intracranial pressure and brain water content, indicating modulation of the elastic modulus by mild hypothermia. An examination with atomic force microscopy (AFM) in a cell injury model in vitro further verified hypothermia-regulated elastic modulus. In this study, we found a novel role of mild hypothermia in modulating the elastic modulus of the injured brain, resulting in the promotion of neural lineage specification of UCMSCs, which suggested that the combination of mild hypothermia had more advantages in cell-based therapy after TBI.

Extracellular Matrix Regulation of Stem Cell Fate

The extracellular matrix (ECM) presents a complex myriad of biochemical and physical cues in the stem cell niche and is able to modulate stem cell fate and function. This review summarizes engineering approaches that have exploited natural and synthetic biomaterials to understand ECM regulation of stem cell fate. Specifically, we demonstrate how these studies have advanced our understanding of vascular maturation and mesenchymal lineage specification. ECM mechanics have emerged as a critical cue in stem cell lineage specification. With the introduction of mechanically dynamic materials, which mirror the non-linear elastic behavior of natural matrices, our understanding of differentiation behavior has evolved. While studies using conventional culture employing rigid, two-dimensional surfaces have greatly advanced our understanding of stem cell differentiation, they overlook the complexity of ECM in the stem cell environment. Implementing defined analogs, through material science and tissue engineering approaches, will allow us to mirror the dynamic nature of ECM and fully elucidate how stem cells differentiate.

From Flies to Mice: The Emerging Role of Non-Canonical PRC1 Members in Mammalian Development

Originally two types of Polycomb Repressive Complexes (PRCs) were described, canonical PRC1 (cPRC1) and PRC2. Recently, a versatile set of complexes were identified and brought up several dilemmas in PRC mediated repression. These new class of complexes were named as non-canonical PRC1s (ncPRC1s). Both cPRC1s and ncPRC1s contain Ring finger protein (RING1, RNF2) and Polycomb group ring finger catalytic (PCGF) core, but in ncPRCs, RING and YY1 binding protein (RYBP), or YY1 associated factor 2 (YAF2), replaces the Chromobox (CBX) and Polyhomeotic (PHC) subunits found in cPRC1s. Additionally, ncPRC1 subunits can associate with versatile accessory proteins, which determine their functional specificity. Homozygous null mutations of the ncPRC members in mice are often lethal or cause infertility, which underlines their essential functions in mammalian development. In this review, we summarize the mouse knockout phenotypes of subunits of the six major ncPRCs. We highlight several aspects of their discovery from fly to mice and emerging role in target recognition, embryogenesis and cell-fate decision making. We gathered data from stem cell mediated in vitro differentiation assays and genetically engineered mouse models. Accumulating evidence suggests that ncPRC1s play profound role in mammalian embryogenesis by regulating gene expression during lineage specification of pluripotent stem cells.

Scaffold Stiffness at Microscale Directs Stem Cell Lineage Specification

Scaffold Stiffness at Microscale Directs Stem Cell Lineage Specification

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis.

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling.

Loss of RUNX1 is associated with aggressive lung adenocarcinomas.

The mammalian runt-related factor 1 (RUNX1) is a master transcription factor that regulates lineage specification of hematopoietic stem cells. RUNX1 translocations result in the development of myeloid leukemias. Recently, RUNX1 has been implicated as a tumor suppressor in other cancers. We postulated RUNX1 expression may be associated with lung adenocarcinoma etiology and/or progression. We evaluated the association of RUNX1 mRNA expression with overall survival data from The Cancer Genome Atlas (TCGA), a publically available database. Compared to high expression levels, Low RUNX1 levels from lung adenocarcinomas were associated with a worse overall survival (Hazard Ratio = 2.014 (1.042-3.730 95% confidence interval), log-rank p = 0.035) compared to those that expressed high RUNX1 levels. Further immunohistochemical examination of 85 surgical specimens resected at the University of Vermont Medical Center identified that low RUNX1 protein expression was associated with larger tumors (p = 0.038). Gene expression network analysis was performed on the same subset of TCGA cases that demonstrated differential survival by RUNX1 expression. This analysis, which reveals regulatory relationships, showed that reduced RUNX1 levels were closely linked to upregulation of the transcription factor E2F1. To interrogate this relationship, RUNX1 was depleted in a lung cancer cell line that expresses high levels of RUNX1. Loss of RUNX1 resulted in enhanced proliferation, migration, and invasion. RUNX1 depletion also resulted in increased mRNA expression of E2F1 and multiple E2F1 target genes. Our data implicate loss of RUNX1 as driver of lung adenocarcinoma aggression, potentially through deregulation of the E2F1 pathway.

SCARA5 plays a critical role in the commitment of mesenchymal stem cells to adipogenesis

Mesenchymal stem cells have the capacity to give rise to multiple cell types, such as adipocytes, osteoblasts, chondrocytes, and myocytes. However, the molecular events responsible for the lineage specification and differentiation of mesenchymal stem cells remain unclear. Using gene expression profile studies, we determined that Scavenger receptor class A, member 5 (SCARA5) is a novel mediator of adipocyte commitment. SCARA5 was expressed at a higher level in committed A33 preadipocyte cells compared to C3H10T1/2 pluripotent stem cells. Gain- and loss-of-function studies likewise revealed that SCARA5 acts as a mediator of adipocyte commitment and differentiation in both A33 and C3H10T1/2 cells. RNAi-mediated knockdown of SCARA5 in A33 cells markedly inhibited the adipogenic potential, whereas overexpression of SCARA5 enhanced adipocyte differentiation in C3H10T1/2 cells. We also demonstrated that the focal adhesion kinase (FAK) and ERK signaling pathways is associated with the SCARA5-mediated response, thereby modulating adipocyte lineage commitment and adipocyte differentiation. Additionally, glucocorticoids induced the expression of SCARA5 in differentiating adipocytes through glucocorticoids response elements (GRE) in the SCARA5 promoter. Taken together, our study demonstrates that SCARA5 is a positive regulator in adipocyte lineage commitment and early adipogenesis in mesenchymal stem cells.

Microbiota regulates bone marrow mesenchymal stem cell lineage differentiation and immunomodulation

Health is dependent on the homeostasis of both inner and external microenvironments. The microbiota as the external microenvironment plays a critical role in regulation of several organ systems in mammals. However, it is unclear whether the microbiota regulates homeostasis of the skeletal system and bone marrow mesenchymal stem cells (BMMSCs). Here, using a well-established germ-free (GF) mouse model, we show that the microbiota significantly alters the stemness of BMMSCs in comparison to specific-pathogen-free (SPF)-derived BMMSCs. Colonization of GF mice with SPF microbiota (conventionalized (ConvD)) normalizes the proliferation and differentiation abilities of BMMSCs. On the other hand, normal microbiota is required to maintain immunomodulatory properties of BMMSCs through induction of activated T-cell apoptosis and cytokine secretion. GF-derived BMMSCs lose the capacity to ameliorate disease phenotypes in dextran sulfate sodium-induced experimental colitis mice. Mechanistically, single-cell RNA-sequencing analysis shows that ConvD BMMSCs have a similar gene expression pattern to SPF-derived BMMSCs, which have a distinct gene distribution from GF-derived BMMSCs.

Mechanisms of stem cell based cardiac repair-gap junctional signaling promotes the cardiac lineage specification of mesenchymal stem cells

Different subtypes of bone marrow-derived stem cells are characterized by varying functionality and activity after transplantation into the infarcted heart. Improvement of stem cell therapeutics requires deep knowledge about the mechanisms that mediate the benefits of stem cell treatment. Here, we demonstrated that co-transplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) led to enhanced synergistic effects on cardiac remodeling. While HSCs were associated with blood vessel formation, MSCs were found to possess transdifferentiation capacity. This cardiomyogenic plasticity of MSCs was strongly promoted by a gap junction-dependent crosstalk between myocytes and stem cells. The inhibition of cell-cell coupling significantly reduced the expression of the cardiac specific transcription factors NKX2.5 and GATA4. Interestingly, we observed that small non-coding RNAs are exchanged between MSCs and cardiomyocytes in a GJ-dependent manner that might contribute to the transdifferentiation process of MSCs within a cardiac environment. Our results suggest that the predominant mechanism of HSCs contribution to cardiac regeneration is based on their ability to regulate angiogenesis. In contrast, transplanted MSCs have the capability for intercellular communication with surrounding cardiomyocytes, which triggers the intrinsic program of cardiogenic lineage specification of MSCs by providing cardiomyocyte-derived cues.

RUNX3 is required for megakaryocyte-erythroid specification in primary human progenitors

The RUNX family of transcription factors is comprised of three RUNX proteins and a common binding partner, CBFb, that enables DNA binding. These factors regulate stem cell maintenance, lineage specification, and cellular differentiation. In the context of malignancies, RUNX1 and CBFb are the most common targets for fusion and point mutations that give rise to such disease states as familial platelet disorder, acute myelogenous leukemia, and myelodysplastic syndromes. By contrast, RUNX3 dysregulation has been observed in solid tumors. In healthy tissue, RUNX1 is required for the ontogenic switch from primitive to definitive hematopoiesis. In mature organisms, RUNX1 mediates transitions between non-lineage stem cell states, protects against inappropriate stem cell expansion, and plays discrete roles in myeloid differentiation. RUNX2 and RUNX3 have known roles in bone and lymphoid cell specification, respectively. The role of CBFb in lineage specification is not well understood. It is known that erythroid cells down-regulate CBFb during differentiation, but megakaryocytes, a lineage derived from a common progenitor, do not. Therefore, we investigated whether CBFb is required for this lineage bifurcation in primary human progenitors. We found that lentiviral-mediated knock-down of CBFb blocked formation of CD41+ megakaryocytes, but spared formation of glycophorin A+ erythroid cells. When screening knock-downs of each of the RUNX factors, we discovered that only loss of RUNX3 resulted in a similar blockade of megakaryopoiesis. Surprisingly, we observed that loss of RUNX3 also blocked erythropoiesis. These data suggest that RUNX3 plays an integral role in megakaryocyte-erythroid specification, but that CBFb is required only for the megakaryocyte lineage.

Design of Three-Dimensional Scaffolds with Tunable Matrix Stiffness for Directing Stem Cell Lineage Specification: An In Silico Study.

Tissue engineering is a multi-disciplinary area of research bringing together the fields of engineering and life sciences with the aim of fabricating tissue constructs aiding in the regeneration of damaged tissues and organs. Scaffolds play a key role in tissue engineering, acting as the templates for tissue regeneration and guiding the growth of new tissue. The use of stem cells in tissue engineering and regenerative medicine becomes indispensable, especially for applications involving successful long-term restoration of continuously self-renewing tissues, such as skin. The differentiation of stem cells is controlled by a number of cues, of which the nature of the substrate and its innate stiffness plays a vital role in stem cell fate determination. By tuning the substrate stiffness, the differentiation of stem cells can be directed to the desired lineage. Many studies on the effect of substrate stiffness on stem cell differentiation has been reported, but most of those studies are conducted with two-dimensional (2D) substrates. However, the native in vivo tissue microenvironment is three-dimensional (3D) and life science researchers are moving towards 3D cell cultures. Porous 3D scaffolds are widely used by the researchers for 3D cell culture and the properties of such scaffolds affects the cell attachment, proliferation, and differentiation. To this end, the design of porous scaffolds directly influences the stem cell fate determination. There exists a need to have 3D scaffolds with tunable stiffness for directing the differentiation of stem cells into the desired lineage. Given the limited number of biomaterials with all the desired properties, the design of the scaffolds themselves could be used to tune the matrix stiffness. This paper is an in silico study, investigating the effect of various scaffold parameter, namely fiber width, porosity, number of unit cells per layer, number of layers, and material selection, on the matrix stiffness, thereby offering a guideline for design of porous tissue engineering scaffolds with tunable matrix stiffness for directing stem cell lineage specification.

Lineage specification of early embryos and embryonic stem cells at the dawn of enabling technologies

Abstract Establishment of progenitor cell populations and lineage diversity during embryogenesis and the differentiation of pluripotent stem cells is a fascinating and intricate biological process. Conceptually, an understanding of this developmental process provides a framework to integrate stem-cell pluripotency, cell competence and differentiating potential with the activity of extrinsic and intrinsic molecular determinants. The recent advent of enabling technologies of high-resolution transcriptome analysis at the cellular, population and spatial levels proffers the capability of gaining deeper insights into the attributes of the gene regulatory network and molecular signaling in lineage specification and differentiation. In this review, we provide a snapshot of the emerging enabling genomic technologies that contribute to the study of development and stem-cell biology.

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification

ABSTRACTPatterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.

Chromatin dynamics regulate mesenchymal stem cell lineage specification and differentiation to osteogenesis

Multipotent mesenchymal stromal cells (MSCs) are critical for regeneration of multiple tissues. Epigenetic mechanisms are fundamental regulators of lineage specification and cell fate, and as such, we addressed the question of which epigenetic modifications characterize the transition of nascent MSCs to a tissue specific MSC-derived phenotype. By profiling the temporal changes of seven histone marks correlated to gene expression during proliferation, early commitment, matrix deposition, and mineralization stages, we identified distinct epigenetic mechanisms that regulate transcriptional programs necessary for tissue-specific phenotype development. Patterns of stage-specific enrichment of histone modifications revealed distinct modes of repression and activation of gene expression that would not be detected using single endpoint analysis. We discovered that at commitment, H3K27me3 is removed from genes that are upregulated and is not acquired on downregulated genes. Additionally, we found that the absence of H3K4me3 modification at promoters defined a subset of osteoblast-specific upregulated genes, indicating that acquisition of acetyl modifications drive activation of these genes. Significantly, loss or gain of H3K36me3 was the primary predictor of dynamic changes in temporal gene expression. Using unsupervised pattern discovery analysis the signature of osteogenic-related histone modifications identified novel functional cis regulatory modules associated with enhancer regions that control tissue-specific genes. Our work provides a cornerstone to understand the epigenetic regulation of transcriptional programs that are important for MSC lineage commitment and lineage, as well as insights to facilitate MSC-based therapeutic interventions.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.

Neural Crest Stem Cells Can Differentiate to a Cardiomyogenic Lineage with an Ability to Contract in Response to Pulsed Infrared Stimulation.

Cellular cardiomyoplasty has rapidly risen to prominence in the clinic following a myocardial infarction; however, low engraftment of transplanted cells limits the therapeutic benefit to these procedures. Recently, lineage-specific stem cells differentiated into cardiomyocytes have gained much attention to assist in the repair of an injured heart tissue; however, questions regarding the ideal cell source remain. In the present study, we have identified a source that is easy to extract stem cells from and show that the cells present have a high plasticity toward the cardiomyogenic lineage. We focused on the recently discovered neural crest stem cells residing in the periodontal ligament that can be easily obtained through dental procedures. Neural crest stem cells were obtained from human excised third molars and differentiated in culture using a protocol for directed differentiation into cardiomyocytes. Differentiation of cells was assessed through gene expression and immunostaining studies. Optical stimulation using pulsed infrared radiation (IR) (λ = 1863 nm) was delivered to cell aggregates to study their contractile ability. We show that neural crest stem cells can be differentiated to a cardiomyogenic lineage, which was verified through immunostaining and gene expression. We observed a significant increase in cardiomyocyte-specific markers, NK2 homeobox 5 (NKX2.5) and troponin T type 2 (TNNT2), with positive changes in tropomyosin I (TPM1), gap junction protein alpha 1/Cx43 (GJA1/Cx43), and myocyte enhancement factor 2C (MEF2C). Furthermore, we were able to elicit and maintain pulse-by-pulse contractile responses in the derived cells, including in cardiospheres, with pulsed IR delivered at various radiant energies. The contractility in responses to IR could be maintained at different frequencies (0.25-2 Hz) and up to 10-min durations. While these cells did not maintain their contractility following cessation of IR, these cells demonstrated responses to the optical stimuli that are consistent with previous reports. We also found no evidence for irreversible mitochondrial depolarization in these cells following the long duration of infrared stimulation, suggesting the robustness of these cells. Overall, these results suggest the merit of neural crest-derived stem cells for cardiomyogenic applications and a potential cell source for repair that should contribute to efforts to translate cell-based strategies to the clinic.

Influence of Amino Acid Metabolism on Embryonic Stem Cell Function and Differentiation

AbstractEmbryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promise in regenerative medicine because of their ability to differentiate into all 3 primary germ layers. This review describes recent advances in the understanding of the link between the metabolism of ESCs/iPSCs and their maintenance/differentiation in the cell culture setting, with particular emphasis on amino acid (AA) metabolism. ESCs are endowed with unique metabolic features with regard to energy consumption, metabolite flux through particular pathways, and macromolecular synthesis. Therefore, nutrient availability has a strong influence on stem cell growth, self-renewal, and lineage specification, both in vivo and in vitro. Evidence from several laboratories has documented that self-renewal and differentiation of mouse ESCs are critically dependent on proline metabolism, with downstream metabolites possibly serving as signal molecules. Likewise, catabolism of either threonine (mouse) or methionine (human) is required for growth and differentiation of ESCs because these AAs serve as precursors for donor molecules used in histone methylation and acetylation. Epigenetic mechanisms are recognized as critical steps in differentiation, and AA metabolism in ESCs appears to modulate these epigenetic processes. Recent reports also document that, in vitro, the nutrient composition of the culture medium in which ESCs are differentiated into embryoid bodies can influence lineage specification, leading to enrichment of a specific cell type. Although research designed to direct tissue specification of differentiating embryoid bodies in culture is still in its infancy, early results indicate that manipulation of the nutrient milieu can promote or suppress the formation of specific cell lineages.

SWI/SNF-directed stem cell lineage specification: dynamic composition regulates specific stages of skeletal myogenesis.

SWI/SNF chromatin-remodeling complexes are key regulators of the epigenetic modifications that determine whether stem cells maintain pluripotency or commit toward specific lineages through development and during postnatal life. Dynamic combinatorial assembly of multiple variants of SWI/SNF subunits is emerging as the major determinant of the functional versatility of SWI/SNF. Here, we summarize the current knowledge on the structural and functional properties of the alternative SWI/SNF complexes that direct stem cell fate toward skeletal muscle lineage and control distinct stages of skeletal myogenesis. In particular, we will refer to recent evidence pointing to the essential role of two SWI/SNF components not expressed in embryonic stem cells-the catalytic subunit BRM and the structural component BAF60C-whose induction in muscle progenitors coincides with the expansion of their transcriptional repertoire.

Pharmacological Reprogramming of Fibroblasts into Neural Stem Cells by Signaling-Directed Transcriptional Activation

Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small-molecule approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts toward a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study provides an effective chemical approach for generating neural stem cells from mouse fibroblasts and reveals mechanistic insights into underlying reprogramming processes.

Lineage-specific stem cells, signals and asymmetries during stomatal development.

Stomata are dispersed pores found in the epidermis of land plants that facilitate gas exchange for photosynthesis while minimizing water loss. Stomata are formed from progenitor cells, which execute a series of differentiation events and stereotypical cell divisions. The sequential activation of master regulatory basic-helix-loop-helix (bHLH) transcription factors controls the initiation, proliferation and differentiation of stomatal cells. Cell-cell communication mediated by secreted peptides, receptor kinases, and downstream mitogen-activated kinase cascades enforces proper stomatal patterning, and an intrinsic polarity mechanism ensures asymmetric cell divisions. As we review here, recent studies have provided insights into the intrinsic and extrinsic factors that control stomatal development. These findings have also highlighted striking similarities between plants and animals with regards to their mechanisms of specialized cell differentiation.