Stem Cell Lineage Specification Research Articles

A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling.

The mechanisms underlying disparate roles of the canonical Wnt signaling pathway in maintaining self‐renewal or inducing differentiation and lineage specification in embryonic stem cells (ESCs) are not clear. In this study, we provide the first demonstration that self‐renewal versus differentiation of human ESCs (hESCs) in response to Wnt signaling is predominantly determined by a two‐layer regulatory circuit involving β‐catenin, E‐cadherin, PI3K/Akt, and Slug in a time‐dependent manner. Short‐term upregulation of β‐catenin does not lead to the activation of T‐cell factor (TCF)‐eGFP Wnt reporter in hESCs. Instead, it enhances E‐cadherin expression on the cell membrane, thereby enhancing hESC self‐renewal through E‐cadherin‐associated PI3K/Akt signaling. Conversely, long‐term Wnt activation or loss of E‐cadherin intracellular β‐catenin binding domain induces TCF‐eGFP activity and promotes hESC differentiation through β‐catenin‐induced upregulation of Slug. Enhanced expression of Slug leads to a further reduction of E‐cadherin that serves as a β‐catenin “sink” sequestering free cytoplasmic β‐catenin. The formation of such a framework reinforces hESCs to switch from a state of temporal self‐renewal associated with short‐term Wnt/β‐catenin activation to definitive differentiation. Stem Cells 2015;33:1419–1433

Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells

Human embryonic stem cells can be differentiated in vitro into a wide variety of progeny cells by addition of different morphogens and growth factors. Our aim was to monitor the expression pattern of tight junction (TJ) components and various cellular markers during differentiation of stem cell lines toward the hepatic lineage. Human embryonic stem cell lines (HUES1, HUES9) were differentiated into endoderm-like cells, and further differentiated to hepatocyte-like cells. Gene expressions of Oct3/4, Nanog, alpha-fetoprotein, albumin, cytokeratins (CK-7, CK-8, CK-18, CK-19), ATP-binding cassette (ABC) transporters (ABCC2, ABCC7, ABCG2), and various TJ components, including claudin-1, claudin-4, claudin-5, claudin-7, and tricellulin, as well as an extracellular matrix component, agrin were monitored during hepatic differentiation by real-time quantitative PCR. The differentiated cells exhibit epithelial morphology and functional assessments similar to that of hepatocytes. The expression level of stem cell marker genes (Oct3/4 and Nanog) significantly and gradually decreased, while liver-associated genes (alpha-fetoprotein, albumin) reached their highest expression at the end of the differentiation. The endoderm-like cells expressed claudin-1, which declined eventually. The expression levels of cholangiocyte markers including claudin-4, CK-7, CK-19, and agrin gradually increased and reached their highest level at the final stage of differentiation. In contrast, these cells did not express notable level of claudin-7, CK-8 and tricellulin. The marker set used for monitoring differentiation revealed both hepatocyte and cholangiocyte characteristics of the differentiated cells at the final stage. This is the first report describing the expression level changes of various TJ components, and underlining their importance in hepatic differentiation.

Enteroendocrine cells are generated from stem cells through a distinct progenitor in the adult Drosophila posterior midgut.

Functional mature cells are continually replenished by stem cells to maintain tissue homoeostasis. In the adult Drosophila posterior midgut, both terminally differentiated enterocyte (EC) and enteroendocrine (EE) cells are generated from an intestinal stem cell (ISC). However, it is not clear how the two differentiated cells are generated from the ISC. In this study, we found that only ECs are generated through the Su(H)GBE(+) immature progenitor enteroblasts (EBs), whereas EEs are generated from ISCs through a distinct progenitor pre-EE by a novel lineage-tracing system. EEs can be generated from ISCs in three ways: an ISC becoming an EE, an ISC becoming a new ISC and an EE through asymmetric division, or an ISC becoming two EEs through symmetric division. We further identified that the transcriptional factor Prospero (Pros) regulates ISC commitment to EEs. Our data provide direct evidence that different differentiated cells are generated by different modes of stem cell lineage specification within the same tissues.

Near-IR absorbing quantum dots might be usable for growth factor-based differentiation of stem cells

Abstract For stem cell therapy of degenerative diseases, it is necessary to differentiate stem cells into the specific lineage. There are several growth factors which have been used for differentiation of stem cells. Some growth factors can dose-dependently induce differentiation of stem cells so that the increase of growth factor concentration results in production of the higher level of differentiated cells. However, due to the toxicity of some differentiation factors (e.g. retinoic acid), the lower dose of growth factors for the specific lineage differentiation of stem cells is desirable. This paper suggests a new approach in the field of controlled growth factor delivery system using semiconductor nanocrystals; known as quantum dots (QDs). This system contains polymeric microencapsulated growth factor which is conjugated to near infrared (NIR) absorbing QDs. The control release of growth factors from microcapsules in the culture plates can be achieved by irradiation. To modulate growth factor release in response to stem cells needs for differentiation, the intensity and period of irradiation will be controlled. Our hypothesis is based on the fact that QDs can absorb NIR energy and by excitation of electrons and then vibrational relaxation of them become heated when they were irradiated and then release growth factors. We believe that controlled growth factors delivery through the suggested system is an effective method to reduce the amount of growth factors required for differentiation of stem cells.

Redox gene expression of adipose-derived stem cells in response to soft hydrogel

Abstract: Adipose-derived stem cells (ADSCs) showed morphological change to a neuron-like shape, and presented neuronal lineage bias when cultured on a very soft surface. To gain basic insight into gene expression relating to neuronal lineage bias in ADSCs, we examined the correlation between the gene expression levels of neuronal markers and redox proteins that were considered to have a close relation with lineage specification in stem cells. ADSCs were cultured on gelatinous soft hydrogel for 1-2 weeks. The time course changes in the expressions of neuronal genes (TUBB3 and NSE) and redox genes (TRX1, SOD1, SOD2, PRX2, GSTT1, and GSTP1) were monitored using real-time PCR. It was found that the TUBB3 gene had significantly upregulated compared to the control condition for tissue culture polystyrene, indicating that the neuronal gene expression of ADSCs could be achieved on soft hydrogel without the addition of any supplement. The expressions of the TRX1 and SOD1 genes were also observed to have significantly upregulated on the soft hydrogels. The results demonstrate that the upregulation of the neural marker of TUBB3 in the ADSCs correlates well with the upregulation of the redox genes of TRX1 and SOD1, when cultured on appropriate soft hydrogel substrates.

Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL_L, hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

Dgcr8 is Indispensable for Cardiac Lineage Specification in Embryonic Stem Cells

Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.

SWI/SNF Chromatin Remodeling Complex in Regulating Mesenchymal Stem Cell Lineage Specification

Mesenchymal Stem Cells (MSC) can be obtained from various tissues and differentiate into many different lineages, including osteoblasts, adipocytes, chondrocytes, cardiomyocytes, hepatocytes and neural cells both in vivo and in vitro. However, the ability of MSC to differentiate into specific lineages seems to be restricted and requires a deeper understanding of the genetic and epigenetic mechanisms. Epigenetic mechanism refers to a process that regulates heritable alterations in gene expression without changing the DNA sequence. SWI/SNF (SWItch/Sucrose Non-Fermentable), a chromatin-remodeling complex serves as an ideal intervention point for lineage manipulation of MSC. In this review, we discuss the importance of SWI/SNF chromatin remodeling complex in regulating the fate determination of MSC. We propose that selectively manipulation of subunits of SWI/SNF will enhance the lineagespecific differentiation of MSC and improve therapeutic application of MSC.

Origin of cardiomyocytes in the adult heart.

This review article discusses the mechanisms of cardiomyogenesis in the adult heart. They include the re-entry of cardiomyocytes into the cell cycle; dedifferentiation of pre-existing cardiomyocytes, which assume an immature replicating cell phenotype; transdifferentiation of hematopoietic stem cells into cardiomyocytes; and cardiomyocytes derived from activation and lineage specification of resident cardiac stem cells. The recognition of the origin of cardiomyocytes is of critical importance for the development of strategies capable of enhancing the growth response of the myocardium; in fact, cell therapy for the decompensated heart has to be based on the acquisition of this fundamental biological knowledge.

Abstract B49: Role of RAS palmitoyl-acyltransferase DHHC9 in hematopoiesis and NRAS leukemogenesis

Abstract Hyperactivation of RAS is common in human cancer, including hematological malignancies. Most oncogenic RAS proteins have been difficult to target. Identification of alternative targets that block RAS signaling is critical to develop therapies for RAS-related cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Protein palmitoylation regulates the membrane targeting, subcellular trafficking, and functions of proteins. We have previously examined the importance of PTMs in NRAS leukemogenesis and found for the first time that palmitoylation is essential for NRAS leukemogenesis. These studies suggest that targeting RAS palmitoylation may be an effective therapy for cancers involving RAS proteins that rely on palmitoylation for plasma membrane binding. In the previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate RAS palmitoylation. The reaction of protein S-palmitoylation is catalyzed by a family DHHC protein palmitoyl-acyltransferases (PATs). Thus far 24 mamalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9, an X-linked gene), is the ortholog of yeast Ras2 PAT and constitute a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancer. Here we investigate the role of DHHC9 in hematopoiesis and NRAS leukemogenesis in vivo. We found that ectopic expression of DHHC9 in mouse bone marrow cells hinders hematopoiesis, but is incapable of inducing hematological malignancies in mice. We also found that frequency of lineage-specific populations and hematopoietic stem cell phenotype were similar in mice with knockout alleles of ZDHHC9 as that of wild type mice, suggesting that DHHC9 is dispensable for normal hematopoiesis. Expression of oncogenic NRAS in bone marrow cells from ZDHHC9 knockout mice still induced leukemia but the mice survived longer that the wild type control mice. These results suggest that DHHC9 plays a role in the pathogenesis of NRAS-induced leukemia, but it is not the only PATs for RAS palmitoylation. We are identifying additional RAS PATs and developing cancer therapies targeting RAS palmitoylation. Citation Format: Ping Liu, Bo Jiao, Maurine Linder, Ruibao Ren. Role of RAS palmitoyl-acyltransferase DHHC9 in hematopoiesis and NRAS leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B49. doi: 10.1158/1557-3125.RASONC14-B49

Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction.

The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.

A Novel Combination of Homeobox Genes Is Expressed in Mesenchymal Chorionic Stem/Stromal Cells in First Trimester and Term Pregnancies

Human chorionic mesenchymal stem/stromal cells (CMSCs) derived from the placenta are similar to adult tissue-derived MSCs. The aim of this study was to investigate the role of these cells in normal placental development. Transcription factors, particularly members of the homeobox gene family, play crucial roles in maintaining stem cell proliferation and lineage specification in embryonic tissues. In adult tissues and organs, stem cells proliferate at low levels in their niche until they receive cues from the microenvironment to differentiate. The homeobox genes that are expressed in the CMSC niche in placental tissues have not been identified. We used the novel strategy of laser capture microdissection to isolate the stromal component of first trimester villi and excluded the cytotrophoblast and syncytiotrophoblast layers that comprise the outer layer of the chorionic villi. Microarray analysis was then used to screen for homeobox genes in the microdissected tissue. Candidate homeobox genes were selected for further RNA analysis. Immunohistochemistry of candidate genes in first trimester placental villous stromal tissue revealed homeobox genes Meis1, myeloid ectropic viral integration site 1 homolog 2 (MEIS2), H2.0-like Drosophila (HLX), transforming growth factor β-induced factor (TGIF), and distal-less homeobox 5 (DLX5) were expressed in the vascular niche where CMSCs have been shown to reside. Expression of MEIS2, HLX, TGIF, and DLX5 was also detected in scattered stromal cells. Real-time polymerase chain reaction and immunocytochemistry verified expression of MEIS2, HLX, TGIF, and DLX5 homeobox genes in first trimester and term CMSCs. These data suggest a combination of regulatory homeobox genes is expressed in CMSCs from early placental development to term, which may be required for stem cell proliferation and differentiation.

Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells.

Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

Stem Cell Lineage Specification: You Become What You Eat

Nutrient availability and intermediate metabolism are increasingly recognized to govern stem cell behavior. Oburoglu et al. (2014) now demonstrate that glutamine- and glucose-dependent nucleotide synthesis segregate erythroid versus myeloid differentiation during hematopoietic stem cell specification, implicating a metabolism-centric regulation of lineage choices.

Work and Tension: New Evidence that Adherent Cells of the Same Area Do the Same Work Independent of Stiffness and Focal Adhesions

Work and Tension: New Evidence that Adherent Cells of the Same Area Do the Same Work Independent of Stiffness and Focal Adhesions

Glucose and Glutamine Metabolism Regulate Human Hematopoietic Stem Cell Lineage Specification

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

Aging-like Phenotype and Defective Lineage Specification in SIRT1-Deleted Hematopoietic Stem and Progenitor Cells

SummaryAging hematopoietic stem cells (HSCs) exhibit defective lineage specification that is thought to be central to increased incidence of myeloid malignancies and compromised immune competence in the elderly. Mechanisms underlying these age-related defects remain largely unknown. We show that the deacetylase Sirtuin (SIRT)1 is required for homeostatic HSC maintenance. Differentiation of young SIRT1-deleted HSCs is skewed toward myeloid lineage associated with a significant decline in the lymphoid compartment, anemia, and altered expression of associated genes. Combined with HSC accumulation of damaged DNA and expression patterns of age-linked molecules, these have striking overlaps with aged HSCs. We further show that SIRT1 controls HSC homeostasis via the longevity transcription factor FOXO3. These findings suggest that SIRT1 is essential for HSC homeostasis and lineage specification. They also indicate that SIRT1 might contribute to delaying HSC aging.

Rewiring mesenchymal stem cell lineage specification by switching the biophysical microenvironment.

The propensity of stem cells to specify and commit to a particular lineage program is guided by dynamic biophysical and biochemical signals that are temporally regulated. However, most in vitro studies rely on “snapshots” of cell state under static conditions. Here we asked whether changing the biophysical aspects of the substrate could modulate the degree of mesenchymal stem cell (MSC) lineage specification. We chose to explore two diverse differentiation outcomes: MSC osteogenesis and trans-differentiation to neuron-like cells. MSCs were cultured on soft (~0.5 kPa) or stiff (~40 kPa) hydrogels followed by transfer to gels of the opposite stiffness. MSCs on soft gels express elevated neurogenesis markers while MSCs on stiff substrates express elevated osteogenesis markers. Transfer of MSCs from soft to stiff or stiff to soft substrates led to a switch in lineage specification. However, MSCs transferred from stiff to soft substrates maintained elevated osteogenesis markers, suggesting a degree of irreversible activation. Transferring MSCs to micropatterned substrates reveal geometric cues that further modulate lineage reversal. Taken together, this study demonstrates that MSCs remain susceptible to the biophysical properties of the extracellular matrix—even after several weeks of culture—and can redirect lineage specification in response to changes in the microenvironment.

Transient Accumulation of 5-Carboxylcytosine Indicates Involvement of Active Demethylation in Lineage Specification of Neural Stem Cells

5-Methylcytosine (5mC) is an epigenetic modificationinvolved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and invivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.

A multiplex high-throughput gene expression assay to simultaneously detect disease and functional markers in induced pluripotent stem cell-derived retinal pigment epithelium.

There is continuing interest in the development of lineage-specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into "adult-like" cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage-specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained "adult-like" phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high-throughput format. Here, we use a multiplex high-throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell-derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell-derived RPE that was then grown in 96- and 384-well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell-derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription-polymerase chain reaction-based measurements, confirming the ability of this high-throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.