Stem Cell Culture Conditions Research Articles

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering - A Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irriadiated MEF feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium and Bronchiolar Endothelial Growth Media. The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow

Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs.

Effects of tachyplesin I on human U251 glioma stem cells.

Glioblastoma, is one of the most malignant types of intracranial tumor with complex progressive cellular and underlying molecular events. The use of glioma stem cells (GSCs) offers a promising strategy for tumor therapy in the future. Tachyplesin I has been demonstrated to have potential anticancer activity and was first observed in leukocytes. In the present study, the GSC subset was isolated from U251 glioma cells and tachyplesin I was assessed for antitumor activity. As a result, the U251 cells exhibited certain GSC phenotypes, including the expression of stem cell biomarkers CD133 and nestin, when transferred into stem cell culture conditions. The GSCs were grown in an adherent manner in a medium containing serum, while the U251 glioma cells were suspended and cultured in serum‑free medium. Tachyplesin I damaged the structure of GSC and inhibited the culture of GSC spheres in a time and dose‑dependent manner. When tachyplesin I was administered at a concentration of 10‑40 µg/ml, GSC differentiation was induced. GSCs treated with a low dose of tachyplesin I disrupted the plasma membrane and led to a loss of cytoplasmic organelles. These findings indicated that tachyplesin I had an effect on inhibiting tumor stem cells and demonstrated that tachyplesin I inhibited GSCs by disrupting the plasma membranes and inducing GSC differentiation.

Inhibition of glioblastoma malignancy by Lgl1.

lethal giant larvae (lgl) was first identified as a tumor suppressor in Drosophila, where its loss repressed the differentiation and promoted the invasion of neuroblasts, the Drosophila equivalent of the neural stem cell. Recently we have shown that a human homolog of Lgl, Lgl1 (LLGL1), is constitutively phosphorylated and inactivated in glioblastoma cells; this occurs as a downstream consequence of PTEN loss, one of the most frequent genetic events in glioblastoma. Here we have investigated the consequences of this loss of functional Lgl1 in glioblastoma in vivo. We used a doxycycline-inducible system to express a non-phosphorylatable, constitutively active version of Lgl1 (Lgl3SA) in either a glioblastoma cell line or primary glioblastoma cells isolated under neural stem cell culture conditions from patients. In both types of cells, expression of Lgl3SA, but not wild type Lgl1, inhibited cell motility in vitro. Induction of Lgl3SA in intracerebral xenografts markedly reduced the in vivo invasion of primary glioblastoma cells. Lgl3SA expression also induced the differentiation of glioblastoma cells in vitro and in vivo along the neuronal lineage. Thus the central features of Lgl function as a tumor suppressor in Drosophila are conserved in human glioblastoma.

Abstract 4194: Gliomaspheres mimic the hypoxia-Notch signaling axis of glioblastoma tumors

Abstract Background and objectives: Notch signaling plays an oncogenic role during gliomagenesis. Synergism of hypoxia with Notch pathway is known to aggravate different cancers. We studied the association between Notch signaling and hypoxia in human glioblastoma (GBM) samples which are characterized by intratumoral hypoxia. We also carried out in-vitro analysis in U87MG cell line enriched in stem cells under low oxygen conditions. The study aimed at identifying a modular gene subset that might be a characteristic of the Notch pathway response in hypoxic GBM tumors and GBM gliomaspheres; which might assist in individualized therapeutics. Methods: Expression of Notch receptors, Dll/Jag ligands, Hes/Hey targets and hypoxia markers was analyzed in 35 GBMs by q-PCR and immunohistochemistry. Spearman's test, clustering and statistical analyses were applied to assess the correlation between hypoxia and Notch pathway upregulation. Expression analysis was also done in U87MG cells grown in stem cell culture conditions under hypoxia using q-PCR and western blot. Results: Using various statistical analyses, we found HIF-1α to be correlated with the expression of maximum number of Notch pathway genes, followed by PGK1, OPN and VEGF. Well-segregated tumor clusters accounting for differential Notch genes' expression were produced using gene clustering and principal components analysis by ordering the GBMs in decreasing levels of HIF-1α/PGK1. By logistic regression, a five hypoxia marker combination (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for Notch signaling augmentation in GBM. A similar Notch-axis (Notch1/Dll1/Hes1/Hes6/Hey1/Hey2) was activated in U87MG gliomaspheres under hypoxia. Interpretation and conclusions: The hypoxia markers most associated with Notch signaling upregulation in resected GBM were identified. Similar components of the Notch pathway were also found to be activated in hypoxia-exposed gliomaspheres, supporting them as an in-vitro model for studying the perturbations of hypoxia-Notch signaling axis. Citation Format: Khushboo Irshad, Saroj Kant Mohapatra, Harshit Garg, Seema Mishra, Chitra Sarkar, Deepak Gupta, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. Gliomaspheres mimic the hypoxia-Notch signaling axis of glioblastoma tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2014-4194

Abstract 3133: The anti-tumor effects of acetazolamide and sulforaphane on bronchial carcinoids: Preclinical modeling and mechanism

Abstract Background: The prognosis of patients with metastatic bronchial carcinoid remains poor despite current conventional and targeted therapies. Here we evaluated the therapeutic potential of acetazolamide (AZ; a pan carbonic anhydrase inhibitor affecting pH regulation and homeostasis) and sulforaphane (SFN; a natural isothiocyanate compound targeting multiple pathways in cancer cells), for their anti-cancer properties. Methods: In vitro and in vivo studies were conducted on H-727 (typical carcinoid) and H-720 (atypical carcinoid) bronchial carcinoid cell lines. We developed a lung orthotopic bronchial carcinoid tumor xenograft model in NOD/SCID mice. Results: AZ and/or SFN significantly inhibited cell viability and clonogenic capacity in a dose-dependent manner (0-80 μM, 48 hours and 7 days). AZ and/or SFN downregulated phosphoH3, Ki67, EPCAM, CA9, Akt1 and upregulated p21 and Nrf2 proteins compared to controls as confirmed by Western blot. Upregulation of Nrf2 gene expression was confirmed by qPCR. AZ and/or SFN significantly reduced xenograft growth and serotonin content after two weeks treatment. In the lung orthotopic model, MR imaging at 3 Tesla precisely identified growth of H-727 injected cells in the ipsilateral side, and revealed liver metastasis as confirmed by histology. A tumor initiating cell (TIC) fraction isolated under stem cell culture conditions showing significantly enhanced tumorigenicity was also studied. Conclusions: AZ and/or SFN inhibited tumor survival, proliferation, invasiveness, serotonin secretion and tumorigenic potential of bronchial carcinoid cell lines. Since the combination of AZ+SFN was more effective than either single agent, we postulate that AZ potentiates the effects of SFN by inhibiting PI3K/AKT, which could enhance the inhibitory effect of SFN on P21, Ki67 and phosphoH3. Interestingly, Nrf2 upregulation by both AZ and by SFN suggested a potent antioxidant response. The lung orthotopic tumor model of bronchial carcinoids permits study of carcinoid metastatic progression. As the doses used in this study are clinically bioavailable we suggest that AZ and SFN may have promising potential for carcinoid therapy and resolution of the carcinoid syndrome. Citation Format: Reza Bayat Mokhtari, Syed S. Islam, Narges Baluch, Karen Aitken, Sushil Kumar, Hai-Ling Margaret Cheng, Mehrdad Yazdanpanah, Khosrow Adeli, Yuanxiang Zhou, Ernest Cutz, Herman Yeger. The anti-tumor effects of acetazolamide and sulforaphane on bronchial carcinoids: Preclinical modeling and mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2014-3133

Optimization of Culture Conditions for Stem Cells Derived from Human Anterior Cruciate Ligament and Bone Marrow

Tissue engineering with stem cells is a fascinating approach for treating anterior cruciate ligament (ACL) injuries. In our previous study, stem cells isolated from the human anterior cruciate ligament were shown to possess extensive proliferation and differentiation capabilities when treated with specific growth factors. However, optimal culture conditions and the usefulness of fetal bovine serum (FBS) as a growth factor in in vitro culture systems are yet to be determined. In this study, we compared the effects of different culture media containing combinations of various concentrations of FBS and the growth factors basic fibroblastic growth factor (bFGF) and transforming growth factor-β1 (TGF-β1) on the proliferation and differentiation of ligament-derived stem cells (LSCs) and bone marrow mesenchymal stem cells (BMSCs). We found that α-MEM plus 10% FBS and bFGF was able to maintain both LSCs and BMSCs in a relatively undifferentiated state but with lower major extracellular matrix (ECM) component gene expression and protein production, which is beneficial for stem cell expansion. However, the differentiation and proliferation potentials of LSCs and BMSCs were increased when cultured in MesenPRO, a commercially available stem cell medium containing 2% FBS. MesenPRO in conjunction with TGF-β1 had the greatest ability to induce the differentiation of BMSCs and LSCs to ligament fibroblasts, which was evidenced by the highest ligamentous ECM gene expression and protein production. These results indicate that culture media and growth factors play a very important role in the success of tissue engineering. With α-MEM plus 10% FBS and bFGF, rapid proliferation of stem cells can be achieved. In this study, MesenPRO was able to promote differentiation of both LSCs and BMSCs to ligament fibroblasts. Differentiation was further increased by TGF-β1. With increasing understanding of the effects of different culture media and growth factors, manipulation of stem cells in the desired direction for ligament tissue engineering can be achieved.

Bioluminescent Human Thyrospheres Allow Noninvasive Detection of Anaplastic Thyroid Cancer Growth and Metastases In Vivo

We have previously demonstrated that thyrospheres derived from human anaplastic thyroid cancer (ATC) cell lines can reconstitute and sustain tumor growth in vivo. The aim of this study was to use luciferase-expressing thyrospheres to establish a clinically relevant mouse model of ATC that allows noninvasive and sensitive monitoring of tumor progression. Two human ATC cell lines stably transfected with a firefly luciferase gene were used to generate thyrospheres under stem cell culture conditions. Cells were orthotopically implanted into the thyroids of immunodeficient NOD/SCIDIl2rg-/- mice to initiate tumors. Tumor progression and metastasis were evaluated by bioluminescent imaging weekly as well as histologic analysis postmortem. We show that only 100 thyrosphere cells are needed for tumor development, and that tumors can be monitored with bioluminescent imaging as early as 7-14 days after implantation. Subsequent histologic evaluation of tissue sections confirmed characteristics of high-grade malignant neoplasms. This approach offers rapid and highly sensitive noninvasive detection options for the preclinical assessment of novel ATC therapeutics in vivo.

Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells

The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological modulation of calpain activity prevents spheroid formation and causes disassembly of preexisting spheroids into single cells, thereby providing novel strategies for improving suspension culture conditions for human pluripotent stem cells in the future.

Comparison of human platelet lysate and fetal bovine serum for optimal culture conditions of neonatal and adult mesenchymal stem cells

Fetal bovine serum (FBS) has significantly contributed to the large-scale expansion of animal and human mesenchymal stem/stromal cells (MSCs) and the rapid development of cell-based therapeutics; however, it poses several regulatory and species cross-contamination challenges hindering the clinical transition of most products. Serum-free media (SFM) supplemented with several growth factors has been proposed as an alternative approach to FBS; however, custom media development is often needed based on the cell type, source, and species. Also, the high cost of SFM makes it an impractical option for large-scale cell expansion. Hence, there is a pressing need to find a human-based media additive. hPL is derived from human platelets and contains similar growth factors and cytokines found in FBS at comparable levels. It has been previously demonstrated that hPL supports the growth of various cells. The focus of this study was to evaluate the ability of a serum converted hPL that does not require the use of heparin (PL-NH) and standard hPL requiring heparin (PL-H) to support attachment, proliferation, and maintenance of multipotent properties of neonatal and adult MSCs from different tissues at different concentrations of media. Variants of hPL used in this study (COOK HPL™ NH and H) are produced at an industrial scale (minimum lot size of 20 L) with high lot-to-lot consistency and purity. Preliminary results of both versions of hPL at different concentrations (2.5, 5, and 10%) supported cell attachment and proliferation of amniotic-, bone marrow-, adipose-, and cord blood-derived MSCs to comparable levels to FBS at all concentrations. The multipotency of MSCs expanded in hPL was maintained throughout several passages. This study demonstrates the ability of using hPL as an alternative media additive to FBS for large-scale expansion of adult and neonatal stem cells for cell-based therapeutics.

Bright/Arid3A Acts as a Barrier to Somatic Cell Reprogramming through Direct Regulation of Oct4, Sox2, and Nanog

SummaryWe show here that singular loss of the Bright/Arid3A transcription factor leads to reprograming of mouse embryonic fibroblasts (MEFs) and enhancement of standard four-factor (4F) reprogramming. Bright-deficient MEFs bypass senescence and, under standard embryonic stem cell (ESC) culture conditions, spontaneously form clones that in vitro express pluripotency markers, differentiate to all germ lineages, and in vivo form teratomas and chimeric mice. We demonstrate that BRIGHT binds directly to the promoter/enhancer regions of Oct4, Sox2, and Nanog to contribute to their repression in both MEFs and ESCs. Thus, elimination of the BRIGHT barrier may provide an approach for somatic cell reprogramming.

Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

BackgroundThe use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA.ResultsRelative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P < 0.001) and 5-bromo-2'-deoxyuridine (BrdU) incorporation (167 ± 6 and 120 ± 6%, respectively; P < 0.001). Treatment with DEX increased ALP activity compared with control (1,638 ± 38%; P < 0.001). In the absence and presence of Dex, BMP-2 did not alter ALP activity (P > 0.8). Runt-related transcription factor2 expression increased 3-fold (P < 0.001) by d 6 of culture. Osterix expression increased 9-fold (P < 0.05) by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 (P < 0.01); however expression was reduced 4-fold at d 18 (P < 0.01).ConclusionsDexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

Identification of U251 glioma stem cells and their heterogeneous stem-like phenotypes

Glioblastoma, the most common and lethal type of intracranial tumor, is characterized by extensive heterogeneity at the cellular and molecular levels. The discovery of glioma stem cells (GSCs) lends support to a new paradigm in tumor biology. In the present study, we aimed to clarify the validity of using U251 glioma cells as a source of GSC culture and critically evaluate the heterogeneous stem-like phenotypes of these cells when grown under various culture conditions. The findings suggested that U251 cells (U251-Adh, U251-SC-Sph and U251-SC-Adh) showed distinctive growth patterns and self-renewal capacity. The U251 glioma cell line is endowed with certain GSC phenotypes that may be moderately enriched in vitro when transferred into stem cell culture conditions, although this is not sustainable and reproducible in vivo. Notably, glioma cells are plastic in response to their environment. The reversible adaptive plasticity contributes to the GSC heterogeneity, which may lead to the heterogeneity of glioblastoma and the differing responses to current therapies. Therefore, an improved understanding of GSC heterogeneity is urgently required for designing more effective therapies against this highly malignant brain tumor.

Restoring the resting potential brings maturity to induced stem cell–derived human cardiac myocytes

Reprogramming of mature human cells to produce the equivalent of embryonic stem cells offers exciting possibilities for therapeutic tissue repair and artificial organs. A more immediate benefit is the possibility of testing potential new drugs and other therapies in human cells of any type that can easily be created in vitro without the use of human blastocysts and with a minimum of invasive procedures in patients. Fundamental research in embryology lead to the identification of growth factors that could be used to force cultured embryonic stem cells to differentiate into mature adult cells including atrial and ventricular cardiac myocytes. This work grew out of the initial approach of allowing the harvesting and subculturing regions of embryos growing in vitro that showed spontaneous beating. A spectacular advance came in 2006 when Takahashi and Yamanaka 1 showed that mouse or human adult fibroblasts could revert to pluripotent stem cells when exposed to 4 factors: Oct3/4, Sox2, c-Myc, and Klf4 under embryonic stem cell culture conditions. In 2012, Yamanaka received the Nobel Prize in Medicine or Physiology for this work, which he shared with Gurdon, who showed in l962 that the specialization of mature cells can be reversed by transplantation of a frog intestinal cell nucleus into a frog egg. By the time of this award, substantial work by others had shown what additional factors or conditions are needed to cause the so-called induced pluripotent stem (IPS) cells to differentiate into adult cardiac myocytes or other cellular components including vascular and endothelial cells. 2 In the heart, embryonic development and tissue differentiation is accompanied by important changes in the electrical activity that alter the cardiac action potential and underlying ionic currents. While the genome must contain sequence information for all ion channels in the heart and other tissues, the expression of the ion channels varies with the stage of embryonic development and with anatomic location. In the embryonic hearts of vertebrates, the action potential of the immature ventricle is similar to that found in the adult sinoatrial node. Specifically, there is spontaneous diastolic depolarization (the pacemaker potential) with a relatively low maximum diastolic potential in immature atrial and ventricular cells, which disappears during development. The upstroke of the action potential is mediated by calcium channels in the early embryo and by sodium channels in the late embryo and adult. The action potential upstroke becomes increasingly rapid during embryonic development, as demonstrated by Nathan and DeHaan 3 in the chick heart. Mature atrial and ventricular cells have a resting potential that is due to predominant permeability to potassium and is approximately � 90 mV, which is the potassium equilibrium

Silencing of RegIV by shRNA causes the loss of stemness properties of cancer stem cells in MKN45 gastric cancer cells

Regenerating islet-derived family member 4 (RegIV) is overexpressed in several types of tumours, including pancreatic and gastric cancer (GC). However, the role it plays in gastric cancer stem cells (GCSCs) remains unknown. The present study tested the hypothesis that the silencing of RegIV by shRNA in GC cells may cause the loss of their stemness properties, indicating the inhibition of growth, proliferation and increased sensitivity to chemoradiation-induced cell death. MKN45 poorly differentiated human GC cells were propagated as mammospheres in stem cell culture conditions. Mammospheres were identified as CSCs using generally acknowledged CSC markers such as CD44. A panel of 21-nucleotide shRNAs were designed to target RegIV gene expression. Several shRNA constructs were identified that led to significant reduction in RegIV mRNA expression. Furthermore, the stemness properties of control mammospheres and RegIV knockdown mammospheres were compared by tumourigenicity assay in vivo and plate colony formation assay in vitro. Finally, we evaluated the treatment response in both mammospheres which underwent chemoradiation. The knockdown expression of RegIV by shRNA deprived CSCs of their stemness properties and increased the effectiveness of cell killing following chemoradiation. Inhibition of endogenous RegIV expression may be a new therapeutic strategy for human GC.

Development of a multiplexed microfluidic platform for the automated cultivation of embryonic stem cells.

We present a multiplexed platform for a microfabricated stem cell culture device. The modular platform contains all the components to control stem cell culture conditions in an automated fashion. It does not require an incubator during perfusion culture and can be mounted on the stage of an inverted fluorescence microscope for high-frequency imaging of stem cell cultures. A pressure-driven pump provides control over the medium flow rate and offers switching of the flow rates. Flow rates of the pump are characterized for different pressure settings, and a linear correlation between the applied pressure and the flow rate in the cell culture devices is shown. In addition, the pump operates with two culture medium reservoirs, thus enabling the switching of the culture medium on-the-fly during a cell culture experiment. Also, with our platform, the culture medium reservoirs are cooled to prevent medium degradation during long-term experiments. Media temperature is then adjusted to a higher controlled temperature before entering the microfabricated cell culture device. Furthermore, the temperature is regulated in the microfabricated culture devices themselves. Preliminary culture experiments are demonstrated using mouse embryonic stem cells.

Long-Expected New Start.

On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Professor Paul R. Sanberg (Department of Neurosurgery, College of Medicine, University of South Florida, FL, USA), Executive Editor of Cell Medicine, for providing us such an excellent opportunity to publish the data that were presented at the annual meeting of JSOPMB. I also thank Dr. David Eve, Associate Editor of Cell Medicine, for editing our papers in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to newly join this organization. Cell-based therapy is one of the most important medical fields in JSOPMB because this form of therapy can be useful for a wide range of diseases. Addressing the current problem of severe human donor organ shortage for cell therapies is a big challenge. Research on adult and embryonic stem cells and artificial cell development, in addition to the recent and rapidly evolving invention of induced pluripotent stem (iPS) cells, encourages us to address the problems confronting cell transplantation. Therefore, JSOPMB has now importantly focused on regenerative medicine in collaboration with cell biologists. One of the extremely important missions of the annual meeting of JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 400 members and is run under the direction of Dr. Takehide Asano, the current President of JSOPMB. Excellent presentations conducted at the 38th annual meeting of JSOPMB held on November 25–26, 2011, in Miyagi, Japan, under the supervision of Dr. Takashi Kondo (Professor, Department of Thoracic Surgery, Tohoku University, Miyagi, Japan) were selected and given an opportunity to be published in this special issue of Cell Medicine. Nine of these presentations are herein published in this special JSOPMB issue. Teratani and Kobayashi reviewed results from various groups using their transgenic rats for direct visualization of their tissues at the bench level and in cell transplantation research. This included studies on renal and liver injury, parkinsonian models, and nerve injury and islet isolation. Diabetes continued to be a major focus with two review papers evaluating different aspects of islet transplantation. Kataoka and Noguchi reviewed the relationship between ER stress and the pathogenesis of type 1 and type 2 diabetes and the association between islet transplantation and ER stress. Maruyama et al. reviewed autologous islet transplantation. Stem cell research was a major topic of interest. There were two papers regarding pancreatic stem cells. One by Noguchi et al. explored the culture conditions for mouse pancreatic stem cells, while the other by Kuise et al. the isolation efficiency of mouse pancreatic stem cells. In addition, Kasahara et al. reported that protein fractions secreted by mesenchymal stem cells (MSCs) were capable of activating preserved islets. There were three papers regarding hepatocytes. Dosen et al. reported that hepatic ischemic reperfusion injury could be reduced using saline exposed to electron discharge in a rat model. Miyamoto et al. synthesized positively charged magnetic nanoparticles and observed these nanoparticles inside three-dimensional models of HepG2 spheroids, which mimic tissues. Finally, Hsu et al. showed that citrate phosphate dextrose-supplemented Euro-Collins solution improved the recovery of hepatocytes isolated from warm ischemic rat liver. The theme of this JSOPMB issue is “Long-Expected New Start.” The board members and I are looking forward to seeing further progress in JSOPMB in conjunction with Cell Medicine.

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.

Culture Conditions for Mouse Pancreatic Stem Cells

Recently, mouse pancreatic stem cells have been isolated from adult mouse pancreata. However, these pancreatic stem cells could be maintained only under specific culture conditions with lot-limited fetal bovine serum (FBS). For the efficient isolation and maintenance of mouse pancreatic stem cells, it is important to identify culture conditions that can be used independent of the FBS lot. In this study, we evaluated the culture conditions required to maintain mouse pancreatic stem cells. The mouse pancreatic stem cells derived from the pancreas of a newborn mouse, HN#101, were cultured under the following conditions: 1) Dulbecco's modified Eagle's medium (DMEM) with 20% lot-limited FBS, in which mouse pancreatic stem cells could be cultured without changes in morphology and growth activity; 2) complete embryonic stem (ES) cell media; and 3) complete ES cell media on feeder layers of mitomycin C-treated STO cells, which were the same culture conditions used for mouse ES cells. Under culture conditions #1 and #3, the HN#101 cells continued to form a flat "cobblestone" monolayer and continued to divide actively beyond the population doubling level (PDL) 100 without growth inhibition, but this did not occur under culture condition #2. The gene expression profile and differentiated capacity of the HN#101 cells cultured for 2 months under culture condition #3 were similar to those of HN#101 cells at PDL 50. These data suggest that complete ES cell media on feeder layers could be useful for maintaining the undifferentiated state of pancreatic stem cells.

Aldh1L1 is expressed by postnatal neural stem cells in vivo.

The metabolic enzyme for folate, Aldh1L1, has been shown to be expressed robustly in astrocytes of the brain. It is now well accepted that astrocytes in certain regions of the adult brain also serve as neural stem cells. Here, we examined whether Aldh1L1 is also expressed in postnatal neural stem cells. In vitro, cells in neural stem cell culture conditions have robust Aldh1L1 promoter activity. In vivo, in the adult brain, astroctyes in neurogenic regions express Aldh1L1 in a pattern consistent with inclusion in neural stem cells, and analysis of Aldh1L1+ cell transcriptome profiles from neurogenic regions reveal a robust enrichment of known regulators of neurogenesis. Genetic fate mapping with Aldh1L1 BAC Cre animals reveals adult-born neuroblasts of the rostral migratory stream are derived from Aldh1L1 expressing cells, as are sporadic neurons in other regions of the brain. Combining these lines of evidence from transgenic animals, cell culture, transcriptome profiling, and fate mapping, we conclude that Aldh1L1 is also expressed in neural stem cells in the brain. These findings may influence the future design of experiments utilizing Aldh1L1 genetic tools, and also suggest existing Aldh1L1 bacTRAP mice may be of use for further experiments profiling neural stem cells in vivo.