Tissue formation and organ homeostasis are achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin-dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFβ signaling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell-fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms of how cell-fate specification is interconnected to cell-cycle dynamics and provides insight into autonomous circuitries governing tissue self-formation.
Humans ; Smad2 Protein/genetics* ; Smad3 Protein/genetics* ; Cell Differentiation ; Pluripotent Stem Cells/metabolism* ; Signal Transduction ; Octamer Transcription Factor-3/genetics* ; Enhancer of Zeste Homolog 2 Protein/genetics* ; Nanog Homeobox Protein/genetics* ; Phosphorylation

OBJECTIVE: To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC，KLF4，LIN28，NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future. METHODS: Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4（hBMMSCs-OCT4）were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs. RESULTS: Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802，OCT4 vs SOX2: r=0.4981；NANOG vs SOX2: r=0.7426). CONCLUSION Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2.
Bone Marrow ; Humans ; Induced Pluripotent Stem Cells ; Mesenchymal Stem Cells ; Nanog Homeobox Protein ; genetics ; Octamer Transcription Factor-3 ; genetics ; Transcription Factors ; Up-Regulation

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.
Animals ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; Human Embryonic Stem Cells ; metabolism ; Humans ; Mice ; Nanog Homeobox Protein ; biosynthesis ; genetics ; Pluripotent Stem Cells ; metabolism

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was detected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT-4 cells with the lentivirus constructs, GFP (+) cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were constructed. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were (1.83-3.12) ×10(8) IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP(+) cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could significantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulate the expression of NANOG mRNA are obtained.
Adult ; Case-Control Studies ; Cell Line, Tumor ; Female ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Male ; Middle Aged ; Nanog Homeobox Protein ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Young Adult

OBJECTIVETo explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells.

METHODSReal-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected.

RESULTSBoth NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05).

CONCLUSIONNANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.

Apoptosis ; genetics ; Cell Line, Tumor ; Down-Regulation ; Gene Expression ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Nanog Homeobox Protein ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo observe the effect of rapamycin on the MG-63 osteosarcoma cells (OC), osteosarcoma stem cells (OSC) and on mTOR signaling pathway, and explore the feasibility of rapamycin as a novel therapeutic measure in osteosarcoma chemotherapy regimens.

METHODSOC and OSC were cultured in vitro. Immunofluorescence assay was used to detect the expression of Nanog and Oct4 in OC and OSC. OC and OSC were treated with rapamycin in concentrations of 0, 20, 50 and 100 nmol/L. Semi-quantitative PCR and RT-PCR were used to detect the mTOR mRNA and CCK-8 assay was used to detect cell proliferation, and the cell morphology was observed under an inverted microscope.

RESULTSThe cores of MG-63 cellular spheres exhibited embryonic stem cell characteristics such as Nanog and Oct4 expession. The mTOR pathway was activated in the OSC and the expression of mTOR mRNA was higher in OSC (0.761 ± 0.080) than that in OS (0.406 ± 0.090, P < 0.05) by semi-quantitative PCR. RT-PCR showed that the expression of mTOR mRNA was lower in OSCs treated with 100 nmol/L rapamycin (0.961 ± 0.060) than that with 0 nmol/L rapamycin (1.654 ± 0.246, P < 0.05). Cell counting kit-8 (CCK-8) assay showed that the proliferation of OC treated with 20, 50 and 100 nmol/L rapamycin was significantly inhibited, compared with that with 0 nmol/L rapamycin (P < 0.05). Compared with 0 nmol/L rapamycin, the proliferation of OSC treated with 20 and 50 nmol/L rapamycin was not significantly inhibited (P > 0.05), but that with 100 nmol/L rapamycin was significantly inhibited (P < 0.05). The invert microscopic observation revealed that rapamycin inhibited the formation of OSC spheres.

CONCLUSIONSRapamycin can effectively inhibit cell proliferation and the ability of sphere formation of OSCs. It will provide a basis for a novel therapeutic approach in osteosarcoma chemotherapy regimens.

Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplastic Stem Cells ; metabolism ; pathology ; Octamer Transcription Factor-3 ; metabolism ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction ; Sirolimus ; administration & dosage ; pharmacology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVETo investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms.

METHODSBreast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR.

RESULTSTSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres.

CONCLUSIONTSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; metabolism ; pathology ; CD24 Antigen ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Female ; Histone Deacetylase Inhibitors ; administration & dosage ; pharmacology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Hydroxamic Acids ; administration & dosage ; pharmacology ; Nanog Homeobox Protein ; Neoplastic Stem Cells ; metabolism ; pathology ; RNA, Messenger ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

BACKGROUNDKeratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.

METHODSA K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44(+)/CD24(-) as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin) was analyzed with real-time PCR.

RESULTSMuch higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.

CONCLUSIONK-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; Cadherins ; genetics ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Culture Media, Serum-Free ; Female ; Homeodomain Proteins ; genetics ; Humans ; Keratinocytes ; cytology ; Nanog Homeobox Protein ; Neoplasm Proteins ; genetics ; Neoplastic Stem Cells ; cytology ; metabolism ; Octamer Transcription Factor-3 ; genetics ; Real-Time Polymerase Chain Reaction

BACKGROUNDHuman amniotic epithelial cells (HAECs), which have characteristics of both embryonic and pluripotent stem cells, are therefore a candidate in cell therapy without creating legal or ethical problems. In the present study, we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe) genes.

METHODSThe offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene. The doubly transgenic (TG) mice (n = 20) and wild-type (WT) mice (n = 20) were randomly divided into two groups respectively: the transplantation group treated with HAECs and the control group with phosphate buffered saline. Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice. Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively. Immunofluorescence cytochemistry was used to track the survival of HAECs. Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs. High performance liquid chromatography was used to measure acetylcholine in hippocampus. The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining.

RESULTSAmyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months, but not in WT mice. The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection. The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell. Compared with the control group, transplantation of HAECs can not only significantly improve the spatial memory of the TG mice, but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites.

CONCLUSIONSThese results demonstrate that intracerebroventricular transplantation of HAECs can improve the spatial memory of the double TG mice. The higher content of acetylcholine in hippocampus released by more survived cholinergic neurites is one of the causes of this improvement.

Acetylcholine ; metabolism ; Alzheimer Disease ; genetics ; metabolism ; therapy ; Amnion ; cytology ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; Epithelial Cells ; cytology ; transplantation ; Genotype ; Hippocampus ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Memory Disorders ; genetics ; metabolism ; therapy ; Mice ; Mice, Transgenic ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Polymerase Chain Reaction ; Presenilin-1 ; genetics ; metabolism