Duke team completes ten-year study on gene expression in stem cells – Duke Chronicle

How do cells acquire their identities? In hopes of answering this question, a Duke team recently completed a study explaining the expression of stem cells after a decade of research.

Stem cells are found in every living organism, including humans and plants. They are initially unspecialized and can develop into almost any cell type while dividing to produce new cells over time. At this point, theyre faced with a choice: divide to make copies of themselves or create something new.

To explore how stem cells make this decision, the group researched the expression of stem cells in plants by developing a specialized microscope that takes precise photographs through a technique called light sheet microscopy.

Cara Winter, associate research professor in biology, and postdoctoral associate Pablo Szekely began working on the project in the lab of Philip Benfey, former Paul Kramer distinguished professor of biology who passed away last year.

This eventually grew to a collaboration with the California Institute of Technology, which included a microscopist, a computer scientist, graduate students and undergraduate student assistants. Winter emphasized that the project was a collaborative effort.

There's no one person that knows all of the information that you need to know to make the microscope and the project work. So you really have to work together to figure out how to get to the goal, Winter said.

Researchers at the California Institute of Technology visited the Duke lab to help construct the microscope. Compared to traditional imaging, this specialized light sheet microscope caused less toxicity and photobleaching, which allowed the team to image cells for longer. This was key because the microscope needed a long observation period to collect enough data on the cells.

The microscope tracked the changing colors of the root cells, which indicated whether the cell would divide and the type of division that would occur. The researchers then connected this data with the level of proteins in the cells to explore how those proteins were associated with cell division. The level of high-resolution imaging of stem cells over such a significant period has never been done before.

Szekely noted that this was made possible by using machine learning and other technological tools to answer these biological questions.

The study allowed the team to photograph the expression of multiple genes in the context of a living organism and connect that gene expression to cell division. They were also able to use that data to test a model of a gene regulatory network, which allowed them to gain insight into asymmetric cell division and how it interfaces with the cell cycle.

The groups results were published in Nature. The study's findings connect to further research for humans and other animals in cancer therapies, drugs, and other aspects of the cell cycle.

I'm hoping to continue following with the next steps of this project, following up on sort of the ideas that came out of the paper, trying to understand exactly what these two regulators are doing early in the cell cycle, Winter said. I'm hoping to stick with imaging, continuing imaging and developing new imaging technologies to ask questions that couldn't be asked.

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Aseel Ibrahim is a Trinity first-year and a staff reporter for the news department.

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Duke team completes ten-year study on gene expression in stem cells - Duke Chronicle

HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes … – Nature.com

hiPSC culture

hiPSCs were generated from an Asian female HOIL-1L-deficient patient and healthy controls and kindly donated by the Center for iPS Cell Research and Application (Kyoto University, Kyoto, Japan). Patient-specific (HOIL-1L_1, CiRA-j-0154B and HOIL-1L_2, CiRA-j-0154D) and healthy control hiPSCs (Control_1, CiRA-j-1616-A, Asian female volunteer) were established from the peripheral blood mononuclear cells (PBMCs) using episomal vectors containing reprograming factors30. Another control hiPSC line from Asian male (Control_2, 110F5) was established as described previously31. Each cell line stored in liquid nitrogen using STEM-CELLBANKER (Takara, Cat.# 11924) and once thawed in 37C water bath, it was maintained in mTeSR1 medium (Stem Technologies, Cat.# 85,850) as previously reported32. Each cell was stocked at less than 15 passages, and all experimentations were done between 20 and 45 passages. The sequence of RBCK1 gene was confirmed by Sanger sequencing at the beginning of key experiments. Pluripotency of CiRA-j-1616-A, CiRA-j-0154B and CiRA-j-0154D was evaluated by OCT3/4 and NANOG mRNA expression by TaqMan qPCR and pluripotency of control_2 was evaluated by quantitative PCR analysis of Oct 3/4, Sox2, Klf4, and c-Myc using SYBR green. Cells were passaged every 45days at 1:10 or 1:12 ratio using accutase (Nacalai Tesque, Cat.# 12679-54). Dissociated cells were seeded on Matrigel-coated 6-well plates. The medium was supplemented with 5M Y27632 (TOCRIS, Cat.# 1254), a Rho-associated kinase inhibitor, on the first day of each passage. All cell lines were authenticated by their name, checked their sterrility regularly, and monitored of mycoplasma contamination using by PCR kit (Minerva biolabs, Cat.# 11-9025).

CMs were differentiated from hiPSCs using a previously reported protocol33. Briefly, hiPSCs were seeded into a 12-well growth-factor-reduced (GFR) Matrigel-coated plate, grown for 4days at 37C in 5% CO2 and mTeSR1 medium, and allowed to reach 8090% confluency. On day 0 of differentiation, the medium was changed to differentiation media, which was RPMI containing 2% B27 minus insulin supplement (Gibco, Cat.# A18956-01) and 1012M CHIR99021 (Selleck, Cat.# S2924), a GSK3 inhibitor. After incubation for 24h, the medium was replaced with fresh differentiation medium. On day 3, the medium was replaced with differentiation medium containing 5M IWP-2 (TOCRIS, Cat.# 3533), a Wnt inhibitor. On day 5, the medium was replaced with fresh differentiation medium. On day 7, B27 minus insulin was replaced with a B27 supplement (Gibco, Cat.# 17504044). Differentiated hiPSC-CMs were purified in glucose-depleted lactate medium as described previously34.

C2C12 cells were kindly provided by Dr. Yuji Yamanashi (The Institute of Medical Science, The University of Tokyo)35. The growth medium was DMEM/F12 (Sigma-Aldrich, Cat.# D6421) containing 20% FBS, 2mM glutamine (Gibco, Cat.# 25030081), 100 units/mL penicillin, and 100g/mL streptomycin. Cells were incubated at 37C in a humidified incubator containing 5% CO2. Myoblasts were differentiated into myotubes in DMEM/F12 medium containing 2% horse serum (Gibco, Cat.# 16050122, Lot. 1968945)36,37.

Lenti-CRISPR v2 (Addgene, Cat. # 52961), which contains a puromycin resistance gene, carrying a guide RNA oligonucleotide (5-acctcacccttcagtcacgg-3 for Exon 5 of the Hoil-1l gene or 5-acgcagcaccacggcctcgc-3 for Exon 7 of the Hoil-1l gene) was constructed. HEK293T cells were transfected with the plasmids using Lipofectamine 2000 (Thermo Fisher, Cat.# 11668019). Viruses were harvested at 48h after transfection, and the media were filtered through a 0.45m PES filter. C2C12 cells were transduced with the viruses in medium containing 10g/mL polybrene. At 24h after transduction, puromycin selection was started. The selected cells were collected and KO of Hoil-1l was confirmed by Sanger DNA sequencing.

Myotubes differentiated from C2C12 cells on day 5 of differentiation were fixed in 4% paraformaldehyde (PFA) for 1h at 4C, permeabilized in 0.1% Triton X-100 for 10min at room temperature, and blocked in PBS containing 3% skim milk for 1h. Thereafter, myotubes were stained with an anti-MHC antibody (1:200, mouse monoclonal, R&D Systems, Cat.# MAB4470). The fusion index was calculated by dividing the number of nuclei in myotubes by the total number of nuclei in a field of view36. The MHC density was calculated by dividing the area occupied by MHC-positive myotubes by the total area of the field of view. The fusion index and MHC density were reported as averages of at least three fields of view (>500 total nuclei). Three independent experiments were performed for the calculation. For pluripotency marker analysis, undifferentiated hiPSC colonies were fixed in the same way, and fixed cells were stained with mouse anti-Oct3/4 (1:50, Santa Cruz Biotechnology, Cat.# sc5279) and anti-TRA1-81 (1:100, Millipore, Cat.# MAB4381) antibodies. Cells were then incubated with Alexa Fluor-conjugated secondary antibodies (1:1000) overnight at 4C.Nuclei were stained withHoechst 33342(1:1000, Invitrogen, Cat.#H3570). For immunofluorescence microscopy analysis of hiPSC-CMs, size and multinucleation were analyzed after around 5060days of differentiation and mitosis was analyzed after 20days of differentiation. hiPSC-CMs were replated onto GFR Matrigel-coated 24-well dishes, incubated at 37C in 5% CO2 for 72h, fixed in 4% PFA for 1h at 4C, permeabilized in 0.1% Triton X-100 for 10min at room temperature, and blocked in PBS containing 3% skim milk for 1h. Thereafter, hiPSC-CMs were stained with anti-cTnT (1:100, mouse monoclonal, Thermo Fisher, Cat.# MA5-12960) and anti-phospho-histone H3 (Ser10) (1:1000, rabbit monoclonal, Cell Signaling, Cat.# D7N8E) antibodies. After primary antibody treatment, cells were rinsed three times with PBS for 5min at room temperature and then incubated overnightat 4 with secondary antibodies diluted 1:1000 in PBS. Nuclei were stained with Hoechst 33342. For isotype controls, mouse IgG1 isotype (BD Biosciences, Cat.# 554121) and rabbit IgG isotype (BD Biosciences, Cat.# 550875) were used. All immunofluorescence analyses were performed using a BZ-710X microscope (Keyence). The TUNEL assay was performed using a Cell Death Detection Kit (Roche, Cat.# 11684795910) following the manufacturers protocol.

Myotubes at day 5 of differentiation were lysed in M-PER buffer (Thermo Scientific, Cat.# 78501) containing 1protease inhibitor and then incubated on ice. The samples were sonicated on ice for 30s. The lysates were incubated on ice for 10min and then centrifuged at 15,000rpm for 15min. Protein concentrations were determined using the Bradford assay. Thereafter, 30g of protein was loaded onto each lane of 10% SDS-PAGE gels. The membranes were probed with an anti-MHC antibody (1:100, R&D Systems, Cat.# MAB4470) in blocking buffer (5% BSA) at 4C overnight, washed, incubated in secondary antibodies for 1h at room temperature, developed using ECL western blotting substrate (Bio-Rad, Cat.# 1705060), and imaged using the ChemiDoc MP Imaging System (Bio-Rad). The blots were cut prior to hybridization with antibodies, and two replicates were done at the same time for Fig.1B and Supplementary Fig.2C as shown in supplementaryFig.5.

hiPSC-CMs were dissociated on the day of evaluation by incubating them in 0.25% trypsinEDTA for 1015min at 37C. They were fixed in Cytofix/Cytoperm solution (BD Biosciences, Cat.# 554714) for 20min at 4C, washed with BD Perm/Wash buffer (Cat.# 554723), stained with an anti-cTnT antibody (1:200, mouse monoclonal, Thermo Fisher, Cat.# MA5-12960) followed by Alexa Fluor-conjugated secondary antibodies, and analyzed using FACSverse (BD Biosciences). In cell cycle analysis, hiPSC-CMs after 20days of differentiation were gathered. After fixing and washing the hiPSC-CMs as described above, they were stained with an anti-cTnT antibody (1:200) and an anti-Ki67 antibody (1:400, rabbit monoclonal, Cell Signaling Technology, Cat. # 9129) followed by Alexa Fluor-conjugated secondary antibodies, and analyzed using FACSverse. Data were collected from at least 10,000 events. Data with>70% cTnT populations were used for all experimental analyses.

Total RNA was extracted from day 0 to day 7 of myotube differentiation using an RNeasy Mini Kit (Qiagen, Cat.# 74104) according to the manufacturers instructions. qPCR was performed using SYBR Green PCR Master Mix (Takara, Cat.# RR820) on a StepOnePlus system (Thermo Fisher Scientific) with the Ct method. GAPDH was used to standardize gene expression. Total RNA was extracted from hiPSC-CMs at 4560days after differentiation.

Hoil-1l-KO and control mouse embryos were generated as described previously14. Paraffin sections of E10.5 Hoil-1l null/+ and control littermate mouse embryos were deparaffinized and stained with H&E.

RNA was isolated from C2C12 cells using a RNeasy Mini Kit, according to the manufacturers instructions. RNA integrity was measured using an Agilent 2200 TapeStation and RNA Screen Tapes (Agilent Technologies). Sequencing libraries were prepared using a NEBNext Ultra II RNA Library Kit for Illumina (New England Biolabs) with the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs), according to the manufacturers protocol. Prepared libraries were run on an Illumina HiSeq X sequencing platform in 150bp paired-end mode. Sequencing reads were aligned to the GRCm38 mouse genome assembly using STAR (c.2.5.3). Mapped reads were counted for each gene using the GenomonExression pipeline (https://github.com/Genomon-Project/GenomonExpression). Normalization of the read counts of RNA seq data and differential expression analysis were performed using the Bioconductor package DESeq2 (version 1.26.0). Differentially expressed genes with a greater than twofold change and a false discovery rate less than 0.1 were filtered and evaluated. RNA seq data have been deposited with links to BioProject accession number PRJDB17426 in the DDBJ BioProject database.

GSEA was performed using software (version 4.0.3) from the Broad Institute. Normalized expression data obtained from RNA seq were assessed using GSEA software and the Molecular Signature Database (http://www.broad.mit.edu/gsea/). c5 ontology gene sets were used, and a false discovery rate less than 0.01 was considered to be statistically significant. Pathway enrichment analysis using g:Profiler and visualization of enrichment results in an enrichment map were performed using Cytoscape software (version 3.7.2) as described previously38.

Data are shown as meanSEMs, as indicated in the figure legends. All statistical analyses were performed using Welchs t-test with GraphPad Prism (version 9.00, GraphPad Software). P<0.05 was defined as significant.

Use of patient-derived iPSCs was approved by the Ethics Committee of Kyoto University (R0091 and G0687), and written informed consent obtained from the donor (or their guardians) in accordance with the Declaration of Helsinki.

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HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes ... - Nature.com

SCG CELL THERAPY AND A*STAR LAUNCH JOINT LABS WITH COLLABORATION NEARING S$30 MILLION TO … – PR Newswire

SINGAPORE, April 16, 2024 /PRNewswire/ --SCG Cell Therapy (SCG) and the Agency for Science, Technology and Research (A*STAR) announced the launch joint laboratories for cellular immunotherapies. This collaboration, at a combined funding of close to S$30 million supported under Singapore's Research, Innovation and Enterprise 2025 Plan (RIE2025), aims to advance the development of induced pluripotent stem cell (iPSC) technology to produce novel cell therapies that meet Good Manufacturing Practice (GMP) standards. The collaboration will also establish a talent development programme to train the next generation of experts in this field, in accordance with current GMP and regulatory requirements.

The research and application of new technologies are essential for addressing growing healthcare needs and maintaining long-term sustainability. However, turning laboratory innovations into practical clinical solutions poses significant challenges. These often involve developing manufacturing processes, validating analytical methods, and implementing automation and digitalisation to guarantee the stability and scalability of products.

The joint laboratories, established at SCG's GMP facility and A*STAR's research facility, leverage SCG's and A*STAR's proprietary technologies to develop scalable GMP-grade iPSC and therapeutic products. SCG contributes its specialised, automated cell therapy manufacturing technologies, while A*STAR brings its unique monoclonal antibody assets, iPSC banks, and expertise in process scaling and analytics.

This collaboration bridges the expertise between public sector research and development (R&D) and industry, consolidating resources from SCG Cell Therapy and A*STAR's Bioprocessing Technology Institute (BTI) and Institute of Molecular and Cell Biology (IMCB) to advance innovative R&D towards GMP manufacturing. Additionally, it immerses researchers in the rigorously controlled GMP environment, facilitating the progression from research to clinical application.

"Cellular immunotherapies herald a new era of regenerative medicine, offering hope for patients with cancers and other serious illnesses. As a key player in T cell receptor (TCR) T cell therapeutics, SCG has developed in-house cGMP manufacturing capabilities to supply high-quality cell therapy products to patients. Through this first-of-its-kind joint collaboration with A*STAR, we bring together A*STAR's advanced iPSC technology and bioprocessing capabilities with our expertise in GMP cell therapy manufacturing and clinical development, furthering our mission to provide affordable off-the-shelf cell therapy treatment options to patients", said Christy Ma, Chief Strategy Officer of SCG Cell Therapy.

"The discovery of iPSCs has revolutionised regenerative medicine, offering the potential for standardised, off-the-shelf cell therapies. Through this collaboration with SCG Cell Therapy, we aim to accelerate the translation of iPSC research into clinically viable therapies and strengthen Singapore's position as a global leader in cell therapy innovation. By leveraging our complementary expertise and resources, the joint labs will not only advance iPSC technology for scalable, GMP-compliant cell therapy production but also serve as a platform for nurturing the next generation of talent in this transformative field," said Prof Koh Boon Tong, Executive Director, A*STAR's BTI.

About iPSC

Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent". Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.

About SCG Cell Therapy

SCG is a clinical-stage biotechnology company focusing on the development of novel immunotherapies in infections and its associated cancers. The company targets the most common cancer-causing infections: helicobacter pylori, human papillomavirus, and hepatitis B, and develops a broad and unique pipeline against infections and to prevent and cure its associated cancers. Established and headquartered in Singapore, SCG combines regional advantages in Singapore, China and Germany, covering the entire value chain from innovative drug research and discovery, manufacturing, clinical development and commercialization. For more information about SCG, please visit us at http://www.scgcell.com.

About the Agency for Science, Technology and Research (A*STAR)

The Agency for Science, Technology and Research (A*STAR) is Singapore's lead public sector R&D agency. Through open innovation, we collaborate with our partners in both the public and private sectors to benefit the economy and society. As a Science and Technology Organisation, A*STAR bridges the gap between academia and industry. Our research creates economic growth and jobs for Singapore, and enhances lives by improving societal outcomes in healthcare, urban living, and sustainability. A*STAR plays a key role in nurturing scientific talent and leaders for the wider research community and industry. A*STAR's R&D activities span biomedical sciences to physical sciences and engineering, with research entities primarily located in Biopolis and Fusionopolis. For ongoing news, visit http://www.a-star.edu.sg.

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Original post:
SCG CELL THERAPY AND A*STAR LAUNCH JOINT LABS WITH COLLABORATION NEARING S$30 MILLION TO ... - PR Newswire