Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance

AbstractPluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

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Differentiation of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells into Steroid-Producing Cells

Endocrinology ◽

10.1210/en.2012-1060 ◽

2012 ◽

Vol 153 (9) ◽

pp. 4336-4345 ◽

Cited By ~ 33

Author(s):

Takuhiro Sonoyama ◽

Masakatsu Sone ◽

Kyoko Honda ◽

Daisuke Taura ◽

Katsutoshi Kojima ◽

...

Keyword(s):

Stem Cells ◽

Regulatory Protein ◽

Embryonic Stem ◽

Ips Cells ◽

Steroidogenic Acute Regulatory Protein ◽

Steroidogenic Factor 1 ◽

Differentiated Cells ◽

Steroidogenic Acute Regulatory ◽

Induced Pluripotent ◽

Mesodermal Lineage

Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3′-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3′,5′-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells.

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Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis

Cellular Physiology and Biochemistry ◽

10.1159/000495644 ◽

2018 ◽

Vol 51 (4) ◽

pp. 1533-1543

Author(s):

Jian Hou ◽

Yan Long ◽

Bo Hu ◽

Shaojie Huang ◽

Guangtao Xu ◽

...

Keyword(s):

Flow Cytometry ◽

Growth Factor ◽

Ips Cells ◽

Glycogen Storage ◽

Hepatic Cell ◽

Hepatic Differentiation ◽

Differentiated Cells ◽

Urea Production ◽

Cell Induction ◽

H1 Cells

Background/Aims: Hepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro. Methods: Human embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells. Results: All differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group. Conclusion: Retaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis.

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Determinants of stem cell enrichment in healthy tissues and tumors: implications for non-genetic drug resistance

10.1101/663146 ◽

2019 ◽

Author(s):

Lora D. Weiss ◽

P. van den Driessche ◽

John S. Lowengrub ◽

Dominik Wodarz ◽

Natalia L. Komarova

Keyword(s):

Drug Resistance ◽

Stem Cell ◽

Genetic Resistance ◽

Therapy Resistance ◽

Resistance Mechanisms ◽

Resistance Mutations ◽

Regulatory Processes ◽

Differentiated Cells ◽

Somatic Evolution ◽

Cell Fractions

AbstractDrug resistance is a major challenge for cancer therapy. While resistance mutations are often the focus of investigation, non-genetic resistance mechanisms are also important. One such mechanism is the presence of relatively high fractions of cancer stem cells (CSCs), which have reduced susceptibility to chemotherapy, radiation, and targeted treatments compared to more differentiated cells. The reasons for high CSC fractions (CSC enrichment) are not well understood. Previous experimental and mathematical modeling work identified a particular feedback loop in tumors that can promote CSC enrichment. Here, we use mathematical models of hierarchically structured cell populations to build on this work and to provide a comprehensive analysis of how different feedback regulatory processes that might partially operate in tumors can influence the stem cell fractions during somatic evolution of healthy tissue or during tumor growth. We find that depending on the particular feedback loops that are present, CSC fractions can increase or decrease. We define characteristics of the feedback mechanisms that are required for CSC enrichment to occur, and show how the magnitude of enrichment is determined by parameters. In particular, enrichment requires a reduction in division rates or an increase in death rates with higher population sizes, and the feedback mediators that achieve this can be secreted by either CSCs or by more differentiated cells. The extent of enrichment is determined by the death rate of CSCs, the probability of CSC self-renewal, and by the strength of feedback on cell divisions. Defining these characteristics can guide experimental approaches that aim to screen for and identify feedback mediators that can promote CSC enrichment in specific cancers, which in turn can help understand and overcome the phenomenon of CSC-based therapy resistance.

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PI3K/AKT inhibition in tumor propagating cells of DLBCL reverses R-CHOP resistance by destabilizing SOX2

10.1101/657445 ◽

2019 ◽

Author(s):

Jianfeng Chen ◽

Xiaowen Ge ◽

Wei Zhang ◽

Peipei Ding ◽

Yiqun Du ◽

...

Keyword(s):

Drug Resistance ◽

B Cell ◽

B Cell Lymphoma ◽

Differentiation Therapy ◽

Chop Regimen ◽

Resistance Development ◽

Differentiated Cells ◽

Large B Cell Lymphoma ◽

Germinal Center B Cell ◽

Chemical Inhibition

AbstractDrug resistance is a major obstacle for the success of conventional anticancer therapy, and the development of drug resistance is at least partly attributed to tumor propagating cells (TPCs). Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen. We found that the TPC proportion was remarkably increased in resistant germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL subtypes. Elevated SOX2 was the determinant for resistance development, and SOX2 was phosphorylated by activated PI3K/AKT1 signaling, thus preventing ubiquitin-mediated SOX2 degradation. Furthermore, multiple factors, including BCR, integrins, chemokines and FGFR1/2 signaling, regulated PI3K/AKT1 activation. CDK6 in the GCB subtype and FGFR1/2 in the ABC subtype were SOX2 targets in the PI3K/AKT1 pathway. Chemical inhibition of PI3K/AKT1 in both subtypes, CDK6 in the GCB subtype, and FGFR1/2 in the ABC subtype significantly enhanced the susceptibility of resistant cells to CHO treatment. More importantly, PI3K and FGFR1/2 inhibitors but not a CDK6 inhibitor effectively suppressed the tumor growth of R-CHO-resistant DLBCL cells, most likely by converting TPCs to chemo-sensitive differentiated cells. Therefore, this pro-differentiation therapy against TPCs warrants further study in clinical trials for the treatment of resistant DLBCL.

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HUMAN INDUCED PLURIPOTENT STEM CELL REGION DETECTION IN BRIGHT-FIELD MICROSCOPY IMAGES USING CONVOLUTIONAL NEURAL NETWORKS

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237219500091 ◽

2019 ◽

Vol 31 (02) ◽

pp. 1950009 ◽

Cited By ~ 3

Author(s):

Yuan-Hsiang Chang ◽

Kuniya Abe ◽

Hideo Yokota ◽

Kazuhiro Sudo ◽

Yukio Nakamura ◽

...

Keyword(s):

Regenerative Medicine ◽

Ips Cells ◽

Patient Specific ◽

Specific Cell ◽

Bright Field ◽

Differentiated Cells ◽

Number Of Patients ◽

Bright Field Microscopy ◽

Induced Pluripotent ◽

Microscopy Images

Human induced pluripotent stem (iPS) cells represent an ideal source for patient specific cell-based regenerative medicine. For practical uses of iPS cells, large-scale, cost- and time-effective production of fully reprogrammed iPS cells from a number of patients should be achieved. To achieve this goal, culture protocols for inducing iPS cells as well as methods for selecting fully reprogrammed iPS cells in a mixture of cells which are still in reprogramming and non-iPS differentiated cells, should be improved. This paper proposes a convolutional neural network (CNN) structure to classify a bright-field microscopy image as respective probability images. Each probability image represents regions of differentiated cells, fully reprogrammed iPS cells or cells still in reprogramming, respectively. The CNN classifier was trained by multiple types of image patches which represent differentiated, reprogramming and reprogrammed iPS cells, etc. Classification of an image containing the confirmed iPS cells by the trained CNN classifier shows that high classification accuracy can be achieved. Classifications of sets of time-lapse microscopy images show that growth and transition from CD34[Formula: see text] human cord blood cells through reprogramming to reprogrammed iPS cells can be visualized and quantitatively analyzed by the output time-series probability images. These experiment results show our CNN structure yields a potential tool to detect the differentiated cells that possibly undergo reprogramming to iPS cells for screening reagents or culture conditions in human iPS induction, and ultimately further understand the ideal culturing conditions for practical use in regenerative medicine.

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Speciation of Intracellular Zn, Fe and Cu within both iPS Cells and Differentiated Cells Using HPLC Coupled to ICP-MS

Journal of Analytical & Bioanalytical Techniques ◽

10.4172/2155-9872.1000295 ◽

2016 ◽

Vol 7 (1) ◽

Keyword(s):

Ips Cells ◽

Differentiated Cells ◽

Icp Ms

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Motility of leukemic lymphocytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159382 ◽

1990 ◽

Vol 48 (3) ◽

pp. 368-369

Author(s):

Manoj Raje ◽

Karvita B. Ahluwalia

Keyword(s):

Quantitative Data ◽

Laser Doppler Velocimetry ◽

Acute Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Cellular Space ◽

Differentiated Cells ◽

Poorly Differentiated ◽

Solid Tissues ◽

Well Differentiated ◽

Reduced Mobility

In Acute Lymphocytic Leukemia motility of lymphocytes is associated with dissemination of malignancy and establishment of metastatic foci. Normal and leukemic lymphocytes in circulation reach solid tissues where due to in adequate perfusion some cells get trapped among tissue spaces. Although normal lymphocytes reenter into circulation leukemic lymphocytes are thought to remain entrapped owing to reduced mobility and form secondary metastasis. Cell surface, transmembrane interactions, cytoskeleton and level of cell differentiation are implicated in lymphocyte mobility. An attempt has been made to correlate ultrastructural information with quantitative data obtained by Laser Doppler Velocimetry (LDV). TEM of normal & leukemic lymphocytes revealed heterogeneity in cell populations ranging from well differentiated (Fig. 1) to poorly differentiated cells (Fig. 2). Unlike other cells, surface extensions in differentiated lymphocytes appear to originate by extrusion of large vesicles in to extra cellular space (Fig. 3). This results in persistent unevenness on lymphocyte surface which occurs due to a phenomenon different from that producing surface extensions in other cells.

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