Category Archives: Embryonic Stem Cells

Embryo Patrol. Artificial Embryos Are Not Human Babies | by Karen Marie Shelton | ILLUMINATION-Curated | Jan, 2024 – Medium

Artificial Embryos Are Not Human Babies Didactic Model of Human Embryonic Development Wagner Souza Esilva Wikimedia

Artificial embryos are not human. Theyre simply a cluster of cells. To be legally human, they must meet the definition of an in vitro fertilized human ovum.

As of the end of 2023, artificial embryos couldnt be successfully implanted into mammals or humans. They couldnt lead to pregnancies, and there is no plan for that to happen in the future.

Synthetic embryos utilize stem cells groundbreakingly, sidestepping the need for sperm or eggs. Ongoing breakthroughs might eventually aid research into genetic disorders and improve babies health, including reducing the risk of problem pregnancies and miscarriages.

Artificial embryos are not related to in vitro fertilization (IVF), which can lead to a human pregnancy.

The term is misleading. These structures arent really synthetic, nor are they exactly embryos. But theyre similar. They are tiny balls of cells arising from a sperm fertilizing an egg but created from stem cells grown in the lab.

Synthetic human embryos, or SHEEFs (synthetic human entities with embryo-like features), are created from very early (actually pre-embryonic) zygotic cells called stem cells.

The stem cells are called pluripotent because they have the potential to develop into almost every cell of the body.

The lab-created embryos are not connected to a beating heart or a brain. They do include cells that would typically go on to form a version of a placenta, yolk sac, and embryo itself.

The model embryos, which resemble human versions, recreate the earliest stages of human development. They could provide a crucial window into genetic disorders and the underlying biological causes of recurrent miscarriage.

Robin Lovell-Badge, headent head of stem cell biology and developmental genetics at Francis Crick Institute in London, reported project advancements. She explained weve cultivated embryos to a specific stage just beyond what is equivalent to 14 days of development for a natural embryo.

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Embryo Patrol. Artificial Embryos Are Not Human Babies | by Karen Marie Shelton | ILLUMINATION-Curated | Jan, 2024 - Medium

Clinical applications of stem cell-derived exosomes | Signal Transduction and Targeted Therapy – Nature.com

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Clinical applications of stem cell-derived exosomes | Signal Transduction and Targeted Therapy - Nature.com

Developing in-vivo chimeric lungs with pluripotent stem cells – Drug Target Review

Reverse-blastocyst complementation elucidates the conditions required to form lungs in rat-mouse chimeric models.

Researchers from the Nara Institute of Science and Technology (NAIST) have used the reverse-blastocyst complementation (rBC) method to understand the conditions required to form lungs in rat-mouse chimeric models. They also used the tetraploid-based organ complementation (TOC) method to create a rat-derived lung in their mouse model.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of global deaths. The pathogenesis of COPD is based on the innate and adaptive inflammatory immune response to the inhalation of toxic particles and gases. Although tobacco smoking is the primary cause of this inhalation injury, many other environmental and occupational exposures contribute to the pathology of COPD.1

Lung transplantation is the only viable treatment option, yet finding suitable lung donors is difficult. However, regenerative medicine is advancing the development of lungs from pluripotent stem cells (PSCs) using interspecies animal models. Current expectations for realisation of the promise of PSCs are at the highest they have ever been. However, there are many challenges that need to be addressed in order to bring PSC technology within the grasp of many more patients. Three particular challenges are: tumorigenicity, immunogenicity, and heterogeneity.2

PSCs and embryonic stem cells (ESCs) from one species can be injected into blastocytes, the cluster of diving cells made by a fertilised egg, in a biological technique named blastocyst complementation to create interspecies chimeric animals. This has enabled successful regeneration of the heart, pancreas and kidney in rat-mouse chimeras, but functional lung formation remains to be achieved successfully in vitro due to the complex three-dimensional (3D) structures and multiple cell types needed. This has warranted more research into the viable conditions required to generate PSC-derived organs.

Fibroblast growth factors (FGFs)are polypeptides with various biological activities bothin vivoandin vitro.3 The fibroblast growth factor 10 (Fgf10) and its interaction with the Fgf receptor isoform IIIb (Fgfr2b) in the lungs are essential for lung development. The rBC method in the new study involved injecting mutant ESCs which fail to show lung formation into wild-type (WT) embryos. This allowed for efficient detection of mutant PSCs in the recipient tissue, aiding the determination of the conditions necessary for successful lung formation in the organ-deficient animal.

The team, led by Dr Shunsuke Yuri and Dr Ayako Isotani, discovered that WT ESCs provide uniform contributions across target and non-target organs in the chimeras. This helped to ascertain that a particular number of WT or normal cells are required to overcome the lung development failure in Fgf10-deficient or Fgfr2b-deficient animals.

Having this understanding enabled them to produce rat-derived lungs in the Fgfr2b-deficient mouse embryos with the TOC method, without the requirement of producing a mutant mouse line. Dr Yuri said: Interestingly, we found that the rat epithelial cells conserved intrinsic species-specific timing in the interspecies model, resulting in an underdeveloped lung. Consequently, these lungs remained nonfunctional post-birth.

The studys findings identified the factors required for the successful generation of functional lungs in rat-mouse interspecies chimeras, as well as the issues to overcome. Dr Yuri concluded: We believe that our study makes an important contribution to the literature by presenting a faster and more efficient method of exploring blastocyst complementation.

These novel results can significantly advance the progress toward developingin-vivochimeric lungs for the purpose of transplantation, which could transform the practical application of regenerative medicine.

This study was published in Development.

1 Hogg JC, Timens W. The Pathology of Chronic Obstructive Pulmonary Disease. Annual Review of Pathology: Mechanisms of Disease. 2008 October 27 [2024 January 5]; 4:435-459. Available from: https://doi.org/10.1146/annurev.pathol.4.110807.092145

2 Yamanaka S. Pluripotent Stem Cell-Based Cell Therapy Promise and Challenges. Cell Stem Cell. 2020 October 1 [2024 January 5]; 27(4):523-31. Available from: https://doi.org/10.1016/j.stem.2020.09.014

3 Birnbaum D, Coulier F, Emoto H, Itoh N, Mattei MG, Tagashira S. Structure and Expression of Human Fibroblast Growth Factor-10*. Journal of Biological Chemistry. 1997 September [2024 January 2024]; 272(37):23191-4. Available from: https://doi.org/10.1074/jbc.272.37.23191

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Developing in-vivo chimeric lungs with pluripotent stem cells - Drug Target Review

No, Rep. Steve Scalise Didn’t Vote Against Stem Cell Research From Which He Is Now Benefiting – Yahoo News

A long-dormant medical controversy was revived last week following an announcement from House Majority Leader Steve Scalise. On January 5, his office released a statement indicating that he was undergoing a stem cell transplant as part of his previously announced treatment for multiple myeloma. Hearkening back to the stem cell controversies of the early 2000s, a number of posts emerged onlineincluding a viral Reddit thread and tweet with 54,000 likes and almost 10,000 retweetsaccusing the congressman of hypocrisy for receiving a treatment that he allegedly fought against.

The treatment Scalise is receiving has no relation to the embryonic stem cell research often opposed by pro-life Americans, however, and the congressman has never voted to restrict research into the form of treatment he is receiving.

Scalise first announced his diagnosis of multiple myelomaa rare blood cancerin late August 2023, telling reporters a month later that his body had responded well to his first round of treatment. The good news is the cancer has dropped dramatically because of the success of the chemotherapy, he said in September. The next step for Scalise, as mentioned above, is an autologous stem cell transplant. [Rep. Scalise] is currently undergoing the transplant process, marking a significant milestone in his battle against cancer, his offices January 5 statement read. Once the procedure is completed, he will be recovering under the supervision of his medical team and will work remotely until returning to Washington next month. Scalise is receiving treatment in his home state of Louisiana.

Because multiple myeloma attacks a patients bone marrowan essential tissue for the bodys production of blood cellsstem cell transplants are often used to help replace marrow damaged by the cancer with new and healthy marrow. In a typical autologous stem cell treatment, the kind which Scalise is receiving, a patients own hematopoietic stem cells are extracted and frozen multiple weeks before treatment. These cells used to be extracted from the bone marrow itself, but today most patients are given a growth factor that allows for stem cells to be taken directly from the blood. The patient is then given intensive chemotherapy, often in a single large dose, before receiving a transfusion of his or her own healthy stem cells. It then takes two to three weeks for the transfused stem cells to restore the functionality of the bone marrow, during which patients can be substantially immunocompromised because of their bodies inability to produce the white blood cells necessary for proper immune function.

Unlike embryonic stem cells, which are harvested from early stage human embryos and can take the form of any cell in the body, the hematopoietic stem cells used in the treatment of multiple myeloma are extracted from a patients own body or from a voluntary donor and can develop into only a limited range of blood cells. These are not the type of stem cells that are in an embryo that can become anything, Dr. Marc Braunstein, a hematology and stem cell transplant expert at NYU Langone Health, told The Dispatch Fact Check. These are slightly differentiated stem cells that are destined to become blood cells, but not anything else.

Traditional stem cell therapies are widely accepted and utilized in modern medical practice, unlike the embryonic stem cell research that reached a point of national controversy in the mid-2000s. We can debate the ethics of using embryonic stem cells, Braunstein said, but I think in this case were not talking about that at all. According to Braunstein, even patients who are practicing Jehovahs Witnessa religious group that typically rejects the use of blood transfusionsare often not opposed to autologous stem cell treatments. For those individuals who may be leery about the use of embryonic stem cells, I dont think they would be as concerned with the use of adult hematopoietic stem cells, Braunstein said.

Furthermore, Scalise has not taken any notable votes against stem cell researchembryonic or non-embryonic. Two notable bills intended to advance embryonic stem cell research, the Stem Cell Research Enhancement Act of 2005 and Stem Cell Research Enhancement Act of 2007, passed both the House and Senate, but both were vetoed by then President George W. Bush. These votes occurred prior to Scalise assuming office in May 2008, however, and very little legislative activity involving embryonic stem cell research has happened since.

In September 2020, Scalise co-signed a letter by Mississippi Sen. Roger Wicker calling for an end to taxpayer funded embryonic stem cell research at the National Institutes of Health, but the letter expressed no opposition to non-embryonic stem cell research or treatment. In fact, Scalise voted in favor of the Stem Cell Therapeutic and Research Reauthorization Act of 2010, Stem Cell Therapeutic and Research Reauthorization Act of 2015, and TRANSPLANT Act of 2021, all of which reauthorized a program intended to support patients in need of stem cell transplants.

Asked by The Dispatch Fact Check whether they believed allegations of hypocrisy were unfair, Scalises office declined to comment further, instead saying that the statement on his treatment spoke for itself.

If you have a claim you would like to see us fact check, please send us an email at factcheck@thedispatch.com. If you would like to suggest a correction to this piece or any other Dispatch article, please email corrections@thedispatch.com.

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No, Rep. Steve Scalise Didn't Vote Against Stem Cell Research From Which He Is Now Benefiting - Yahoo News

Embryonic-stem-cell-derived mesenchymal stem cells relieve experimental contact urticaria by regulating the functions … – Nature.com

Animals

All animal experimental procedures were approved by the Dong-A University Medical School Institutional Animal Care and Use Committee (Approval No. DIACUC-21-11) at 22.03.2021. Female BALB/c mice that were 78weeks old were purchased from Orient Bio Inc. (Gyeonggi-do, korea). The classification of experimental groups involves the random assignment of mice of the same age and within a 1g difference in body weight after the acclimatization process. The mice are then housed in a pathogen-free facility at Dong-A University (Busan, Korea). Mice were maintained at Dong-A University facility at 22C1C room temperature, 4060% humidity, on a 12h lightdark cycle (7a.m. to 7p.m.), and given food and water freely, according to institutional guidelines. All experiments were performed under inhalation anesthesia with isoflurane, and mice were euthanized by CO2 inhalation at the end of the experiment. This study adhered to the guidelines set forth by the laboratory animal ethics committee of Dong-A University and the ARRIVE guidelines. To ensure statistical significance, 5 or more mice per group were used, and all experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A University. Inhalational anesthesia using isoflurane was used to induce anesthesia when sacrificing all experimental animals.

Contact urticaria mouse model was induced according to a previously reported method31,32. Mice were initially sensitized by applying 100l of a TMA (trimellitic anhydride; 500mg/ml, Alfa Aesar, Ward Hill, MA, USA) in acetone/olive oil (4:1, v/v) on the shaved hind flank. This sensitization process is essential for inducing an immune response to TMA. Secondary sensitizations were performed on the hind flank to reinforce the immune response. On the 7th and 10th days after the first sensitization, mice sensitized 50l of a TMA solution (250mg/ml) in acetone/olive oil (4:1, v/v). On day 13 after the initial sensitization, contact urticaria (CU) was induced by challenging the ears with 25l of a TMA solution (100mg/ml) dissolved in acetone/olive oil (4:1, v/v). The disease symptoms were assessed by measuring ear thickness, itching, and lesions on the skin. Particularly, skin lesions were evaluated by determining the ratio of the affected area, indicating erythema and edema on a 3 cm2 area of the dorsal skin. The symptom evaluation of experimental animals was evaluated based on all animals without exclusion criteria. For histological analysis, H&E and mast cell staining were conducted. Cellular and molecular analysis involved the use of flow cytometry to assess immune cell activity in mouse lymphoid organs, along with genetic analysis of the lesions. Experimental animals of 10 mice per group were performed by blindly selecting 56 mice for efficient handling of animal-derived flow cytometric and genetic analysis. The corresponding author and one of the first authors (S.Y. Hyun) were aware of the group assignment, and the symptoms, flow cytometry, and other molecular analysis results were evaluated together by multiple blinded co-authors.

For the purposes of in vitro experiments, we used the nave CD3+ T cell isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) to enrich nave CD3+ cells from the spleens of BALB/c mice (6weeks old). All steps were conducted strictly following the manufacturers protocol.

M-MSCs used in this study was provided Mirae Cell Bio (Seoul, Korea). M-MSCs differentiated from H9 hESCs30 were maintained in EGM2-MV medium (Lonza, San Diego, CA, USA) containing supplement Mix (promocell, Heidelberg, Germany) and 50 ug/ml Gentamicin (Gibco, NewYork, USA) in a humidified atmosphere containing 5% CO2 at 37, as previously described33. M-MSCs at less than ten passages were used for in vitro cell culture and in vivo animal experiments. Bone marrow-derived MSCs (BM-MSCs) were maintained in MSCBM medium (Lonza, Basel, Switzerland) containing supplement kit (Lonza) in a humidified incubator at 5% CO2 at 37. Bone marrow-derived mast cells (BMMCs) derived from BALB/c mice were cultured in RPMI 1640 medium containing 2mM L-glutamine, 0.1mM nonessential amino acids, antibiotics, 10% fetal bovine serum (FBS), and IL-3 (10ng/ml; PeproTech Inc., Rocky hill, NJ, USA). After 4weeks,>98% of the cells were verified as BMMCs, as previously described34. Mouse splenic T cells were presorted by CD3 mAb-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by the manufacturer's method. For T cell polarization, splenic nave CD3+ T cells were cultured onto a 24-well plate coated with 1g/ml of anti-CD3 (eBioscience, San Diego, CA, USA) in complete RPMI 1640 medium and supplemented with TH1 reagents [IL-2 (20ng/ml, PeproTech Inc.), IL-12 (20ng/ml, PeproTech Inc.), and anti-IL-4 (10g/ml, Bio X cell, West Lebanon, NH, USA)] or TH2 reagents [IL-2 (20ng/ml, PeproTech Inc.), IL-4 (100ng/ml, PeproTech Inc.), and anti-IFN- (10g/ml, Bio X cell)]. After 48h, the cells were co-cultured with M-MSCs for 24h under polarized conditions. To confirm the regulation of immune cells by M-MSCs in vitro, BMMCs (5.0105 cells/well), splenic T cells (5.0105 cells/well), or polarizing splenic T cells (5.0105 cells/well) were co-cultured with the indicated ratio of M-MSCs for 24h. Co-cultured splenic T cells were identified by flow cytometry analysis. The analysis of polarized T cells was assessed by distinguishing them using the gating strategy as depicted in Supplementary Fig.S1. The ratio of degranulation of BMMCs was analyzed by -hexosaminidase secretion.

After the primary sensitization of the contact urticaria model, M-MSCs were injected subcutaneously into the ear as a single administration on the 10th days or twice on the 10th and 12th days. A primary disease improvement evaluation was performed through single administration or two administrations, and an appropriate cell administration group for disease efficacy was selected. The administration of an equal amount of BM-MSCs and oral administration of cetirizine (50mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. To deplete TGF-, BALB/c mice were intraperitoneally injected with 300g of anti-TGF- mAb (1D11.16.8, Bio X cell) or an isotype-matched control mAb (Bio X cell) twice on days 0 and 3 of M-MSCs administration.

Single-cell suspensions were isolated from the spleen, cervical lymph node (cLN), and ear. Ear tissues were isolated into single cells using the gentleMACS dissociator (Miltenyi Biotec) followed by the manufacturers method. For the detection of intracellular cytokines, the isolated cells were stimulated with PMA (50ng/ml; Sigma-Aldrich), ionomycin (500ng/ml; Sigma-Aldrich), and brefeldin A (3g/ml; eBioscience) for 4h before analysis and a fixation/permeabilization kit were from eBioscience. Before cell surface markers were stained, Fc receptors were blocked with anti-CD16 and anti-CD32 mAbs (2.4G2, BD Biosciences), and conjugated and dead cells were excluded by analysis on the basis of forward and side light scatter parameters and staining with a Zombie NIR Fixable Viability Kit (Biolegend, San Diego, CA, USA). The antibodies against proteins were as follows: Antibodies against CD3 (17A2) and CD8a (53-6.7) were obtained from BioLegend. Antibodies for CD4 (RM4-5), IFN- ((XMG1.2), IL-4 (11B11) were obtained from eBioscience. Antibodies for CD3 (17A2), CD45 (30-F11), and CD127 (A7R34) were obtained from BioLegend. The cells were then analyzed with a NovoCyte flow cytometer (Agilent) and FlowJo version 10 software (Tree Star, Ashland, OR, USA).

BMMCs (5.0105 cells/well) co-cultured with M-MSCs (0.5 to 2106 cells/well) for 24h were sensitized for 4h with Monoclonal dinitrophenol (DNP)-specific IgE (100ng/ml; Sigma). The IgE-primed BMMCs were then stimulated with 50ng/ml of DNP-human serum albumin (DNP-HSA, Sigma-Aldrich) in Tyrode-BSA buffer (20mM Hepes (pH 7.4), 135mM NaCl, 5mM KCl, 1.8mM CaCl2, 1mM MgCl2, 5.6mM glucose, and 0.1% BSA) for 15min in the presence or absence of the M-MSCs.

Degranulation was determined by measuring the release of the granule marker -hexosaminidase as previously described35. The degree of degranulation of BMMCs was expressed as the % of the activity of -hexosaminidase secreted out of the cells compared to the total activity of -hexosaminidase.

M-MSCs were co-cultured with splenic T cells or BMMCs for 24h and then effector cells were removed. M-MSCs were rinsed with PBS and left on ice for 5min to stop the reaction. Total RNA was extracted using AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea), and cDNA was synthesized using AccuPower CycleScript RT PreMix (Bioneer) according to the manufacturers instructions. The PCR reaction was amplified using AccuPower PCR PreMix (Bioneer) and PCR was performed at 95 for 2min, 95 for 20s, 58 for 40s, 72 for 30s, 72 for 5min for 30 cycles. Primers used as follow: human Hgf (forward 5-TCCATGATACCACACGAACACAGC-3, reverse 5-TGCACAGTACTCCCAGCGGGTGTG-3); human Ido1 (forward 5-TTTGCTAAAGGCGCTGTTGG-3, reverse 5-CCTTCATACACCAGACCGTCTGA-3); human Pdl1 (forward 5-TATGGTGGTGCCGACTACAA-3, reverse 5-TGCTTGTCCAGATGACTTCG-3); human Il10 (forward 5-AGACATCAGGGTGGCGACTCTAT-3, reverse 5-GGCTCCCTGGTTTCTCTTCCTAAG-3); human Pge2 (forward 5-ACCATCTACCCCTTCCTTT-3, reverse 5-CCGCTTCCCAGAGGATCT-3); human Tgfb (forward 5-GGGACTATCCACCTGCAAGA -3, reverse 5-CCTCTTGGCGTAGTAGTCG-3); human Gapdh (forward 5-ACCACAGTCCATGCCATCAC-3, reverse 5-TCCACCACCCTGTTGCTGTA-3). Snap-frozen disease-inducing mouse ear tissues were ground to powder. Total RNA isolation and PCR reaction were performed in the same manner as above. Real-time PCR was performed Thermal Cycler Dice Real Time System III TP950 (Takara, Shiga-ken, Japan). Primers used as follow: mouse Il4 (forward 5-ACAGGAGAAGGGACGCCAT-3, reverse 5-GAAGCCCTACAGACGAGCTCA-3); mouse Il6 (forward 5-GAGGATACCACTCCCAACAGACC-3, reverse 5-AAGTGCATCATCGTTGTTCATACA-3); mouse Ifng (forward 5-CAGCAACAGCAAGGCGAAAAAGG-3, reverse 5-TTTCCGCTTCCTGAGGCTGGAT-3); mouse Tnfa (forward 5-AGTGACAAGCCTGTAGCCCACGT -3, reverse 5-CCATCGGCTGGCACCACTAGTT-3); mouse Gapdh (forward 5-CATCACTGCCACCCAGAAGACTG-3, reverse 5-ATGCCAGTGAGCTTCCCGTTCAG-3);

After the induction of contact urticaria in mice, their ear tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline for 24h and then embedded in paraffin. The tissues were dehydrated in a graded ethanol series (70 to 100%), rinsed three times with xylene for 3min each, and then embedded in paraffin. Sections of paraffin-embedded tissues, with a thickness of 6m, were prepared and stained with hematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich) to compare and analyze the degree of cell invasion and epidermal thickness in the tissue. Additionally, sections of tissues with a thickness of 6m were stained with a 1% toluidine blue (Sigma-Aldrich) solution to assess the number of infiltrating mast cells and the degree of degranulation.

The in vitro experiment was repeated three independent times, and the animal experiment was based on five or more animals per group, and if the results of the first experiment were insufficient, the significance was evaluated within a total of 10 animals in the group. The data are presented as the meanstandard error (SEM) from three or more independent experiments for in vitro experiments. Statistical analysis was done by unpaired Student's t-test. One-way analysis of variance (ANOVA) with Tukey's post hoc test was performed for multiple comparisons. Statistical significance (*P<0.05 and **P<0.01) was determined with Prism version 7.0 (GraphPad, San Diego, CA).

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A University(DIACUC-21-11). All animal experiments were performed in accordance with the guidelines and regulationsof the institutional guidelines.

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Embryonic-stem-cell-derived mesenchymal stem cells relieve experimental contact urticaria by regulating the functions ... - Nature.com

Machine learning-based estimation of spatial gene expression pattern during ESC-derived retinal organoid … – Nature.com

CNN architecture and dataset for estimating spatial gene expression patterns

Our model utilizes a CNN that takes a phase-contrast image as input and estimates a fluorescent image as output (Fig.1A). The typical input to a CNN is a two-dimensional (2D) image. This 2D image is passed through several convolution layers, each followed by a nonlinear activation function. The training parameters correspond to the weights of these convolution kernels and the biases. Our network has a U-Net-like architecture27, which is an encoder-decoder structure with skip connections. The embedded features from the encoder are passed through the decoder, which consists of upsampling and convolution layers to increase the resolution of the intermediate feature maps to obtain a fluorescent image as output.

In our model, the ResNet5028 was used as the backbone of the encoder. The size of the input image for the ResNet50 is (3times Htimes W). To use the pre-trained model of the ResNet50, gray-scale phase-contrast images were replicated in the axis of the channel to create three-channel images. At the first layer, a convolution with stride 2 is applied to the input image to generate features of size (64times frac{H}{2}times frac{W}{2}). The ResNet50 has 4 residual blocks and the size of the output features of these blocks are (256times frac{H}{4}times frac{W}{4}), (512times frac{H}{8}times frac{W}{8}), (1024times frac{H}{16}times frac{W}{16}), and (2048times frac{H}{32}times frac{W}{32}), respectively. These features are then concatenated to the decoder to exploit multi-scale information. The output of the decoder is a fluorescent image of size (1times frac{H}{2}times frac{W}{2}). Note that each convolution layer has a batch normalization (BN) layer and a rectified linear unit (ReLU) activation function, except for the final convolution layer, which has a sigmoid activation function to constrain the range of the output values between 0 and 1.

The network was optimized by minimizing the training loss computed on the output and corresponding ground-truth fluorescent images. The combination of mean squared error (MSE) and cosine similarity, which captures structural patterns from the entire image, was used as the training loss.

To train, validate, and test our model, we cultured retinal organoids derived from mouse ESCs using the SFEBq method10. In this culture, a GFP gene was knocked-in under the promoter of a master gene of retinal differentiation, Rax. Using this method, we obtained a dataset of a pair of phase-contrast image and fluorescent image of Rax during retinal differentiation (Fig.1B). Images were captured for 96 organoids at 4.5, 5, 6, 7, and 8days after the start of SFEBq, where each sample was captured as 14 Z-stack images. This resulted in a total of (96times 5times 14=6720) image pairs were obtained. These image pairs were divided into 5880, 420, and 420 samples for training, validation, and test, respectively. 84, 6, and 6 organoids were used for training, validation, and test, respectively; thus, each organoid does not appear in the different datasets. For data augmentation, we randomly flipped the input images vertically and horizontally during training. While the image resolution of both phase-contrast and fluorescent images is (960times 720), the (512times 512) regions where organoids appear were extracted.

To demonstrate our model, we applied it to 420 samples of the test data. As a result, the proposed model successfully estimated the spatial expression patterns of Rax from phase-contrast images during retinal organoid development (Fig.2). During development, multiple optic vesicles are formed through large and complicated deformations (Fig.2A). This process begins with a spherical embryonic body, with some portions of the tissue surface evaginating outward to form hemispherical vesicles, i.e., optic vesicles. Importantly, the resulting morphology of retinal organoids, especially optic vesicles, varies widely29. This process is known to be governed by the expression of the Rax gene (Fig.2B). That is, the Rax gene is gradually expressed in several parts of the tissue surface, so-called eye field, where cells differentiate from neuroepithelium into several types of retinal cells.

Estimated spatial Rax expression patterns during retinal organoid development. (A) Phase-contrast images from day 4.5 to day 8. (B) Captured fluorescent images of Rax as ground-truths. (C) Estimated fluorescent images with our model. (D) Error maps between captured and estimated images. The error metric was a squared error. The organoids in (AD) are identical. Scale bars indicate 200m.

Our model successfully recapitulated the above features of Rax expression (Fig.2C), i.e., the Rax intensity was relatively low and homogenous at days 4.5, 5, 6, and gradually increased around the evaginated tissue regions at days 7 and 8. Remarkably, the regions of high Rax expression were accurately estimated even in organoids with various morphologies. On the other hand, as the Rax intensity increases, especially around the evaginated tissue regions, the error of the estimated image from the ground-truth image increases with time (Fig.2D).

To quantitatively evaluate the accuracy of the estimation, we statistically analyzed the estimation results at each stage. To clarify whether the model can estimate Rax intensity in both samples with high and low Rax expression, each of the ground-truth and estimated fluorescence images was divided into two categories by the coefficient of variation of the foreground pixels in a fluorescent image at day 8 (Fig.3A). The samples in each group were labeled as positive and negative, respectively. For each of these categories, the mean and coefficient of variation of the pixel values were calculated (Fig.3BE). In calculating these values, the phase-contrast images were binarized to obtain foreground and background masks, and then computed using only the foreground pixels and normalized to those of the background pixels.

Statistical analysis of fluorescence at each developmental stage for positive and negative samples. (A) Histogram of coefficient of variation for foreground pixel values of fluorescent images at day 8. (B, C) Means of pixel values in positive and negative samples at each stage for ground-truth (green bars) and estimated fluorescent images (blue bars), respectively. (D, E) Coefficients of variation in positive samples at each stage for both ground-truth (green bars) and estimated fluorescent images (red bars), respectively. (F, G) Plots of ground-truth and estimated pixel values in positive and negative samples at each stage, respectively. Errors are 0% and 25% on the solid and dotted black lines, respectively. Error bars in (BE) indicate standard deviations.

Positive samples showed a gradual increase in mean and intensity over the days passed (Fig.3B). The negative sample, on the other hand, showed relatively low values from the beginning and did not change significantly over the days (Fig.3C). Similarly, the coefficients of variation increased in the positive samples but not in the negative samples (Fig.3D,E). These results indicate that the model successfully estimates the feature of the spatial Rax expression patterns during retinal organoid development, i.e., positive samples gradually increase Rax expressions and their heterogeneity, but negative samples do not. The intensity of the estimated images is relatively lower than the intensity of the ground-truth images in the positive samples and vice versa in the negative samples.

To clarify whether the model is capable to estimate intermediate values of the Rax expression, we analyzed the correlations between ground-truth and estimated values on foreground pixels at each stage, respectively (Fig.3F,G). The results show that in the positive sample (Fig.3F), the distribution of intensities is initially concentrated at low intensities and gradually expands to high intensities as the day progresses, with a wide distribution from low to high intensities. Similarly, in the negative sample, the luminance distribution is initially concentrated at low intensities, but does not expand as much as in the positive sample (Fig.3G). These results indicate that the model successfully estimated the plausible values across all pixel intensities, demonstrating the capability of our method to infer intermediate levels of gene expression. Notably, predicting Rax expression in the organoids at later stages, such as day 8 in our experiments, becomes more feasible for the model due to their characteristic morphologies. These distinct morphologies provide features that can be efficiently extracted by the convolution operators of the model.

To determine whether the estimated Rax expression patterns correspond to tissue morphologies, we quantified the spatial distribution of Rax intensity and the mean curvature along the tissue contour around each optic vesicle (Fig.4). For this analysis, four typical optic vesicles were selected from the positive samples and their curvature and Rax distribution were quantified. In this analysis, tissue contours were extracted and the radius of a circle passing through three points on the tissue contour was calculated as the inverse of the curvature. Moreover, the Rax intensity was measured as the average value along the depth direction from the tissue contour.

Correlation analysis of spatial Rax expression patterns and optic-vesicle morphologies. (A) Phase-contrast images. (B) Captured fluorescent images of Rax as ground-truths. (C) Estimated fluorescent images with our model. (D) Mean curvatures as a function of the distance along the organoid contour. (E) Captured and estimated fluorescent intensities of Rax along the organoid contour. The organoids in (AC) are identical and captured on day 8. The mean curvatures and fluorescence in (D, E) are for the regions indicated by the red line starting from the red dot in (A). Scale bars indicate 200m.

Optic vesicles are hemispherical, with positive curvature at the distal portion and negative curvature at the root (Fig.4A,D). The Rax intensity is continuously distributed around each vesicle, being highest at the distal part and gradually decreasing toward the root (Fig.4B,E). Furthermore, because the test images were taken with a conventional fluorescence microscope, structures above and below the focal plane are included in each image. Therefore, although some images have multiple overlapping vesicles (e.g., samples iii and iv), the model successfully estimated the Rax intensity of the overlapping regions as well.

MSE is commonly used as the training loss for training regression models. In addition to MSE, this model also uses cosine similarity, which can capture structural patterns from the entire image. To analyze the effect of cosine similarity on the estimation accuracy, we tested the model with different weights for both error metrics (Fig.5). The trained models were evaluated with MSE for each test dataset on different days (Fig.5A). The results demonstrated that cosine similarity improved the estimation accuracy at the early and intermediate stages, such as from day 4.5 to day 6. At these stages, the intensity in the differentiated region is weak, making it difficult for the network to capture structural patterns using MSE alone. Cosine similarity, on the other hand, enabled the network to learn the patterns from the weak intensity by calculating the correlation between the normalized ground-truth and the estimated images (Fig.5B). Therefore, our model has the capability to achieve the best estimate at different stages with appropriate weight balancing.

Effects of the balance of training loss on estimation accuracy. (A) Mean squared error at each stage with different hyperparameters, where bold and underlined numbers stand for the best and second best results on each day, respectively. (B) Examples of estimated fluorescent images at days 6 and 8 with different hyperparameters. The MSE of each estimated image is described in the upper left. The results with the lowest MSEs are surrounded by the red boxes. Scale bars indicate 200m.

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Machine learning-based estimation of spatial gene expression pattern during ESC-derived retinal organoid ... - Nature.com

Stem Cell Banking Market Size Revenue Hits $18.04 Billion by 2032 … – GlobeNewswire

Newark, Nov. 20, 2023 (GLOBE NEWSWIRE) -- The Brainy Insights estimates that the USD 7.93 Billion in 2022stem cell banking market will reach USD 18.04 Billion by 2032. As stem cell transplants become more viable therapeutic options, the demand for a reliable and secure source of stem cells has increased significantly. Stem cell banks are critical to the success of these treatments because they provide a secure and dependable means of storing and transferring stem cells for transplantation.

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Report Coverage Details

Key Insight of the Stem Cell Banking Market

Asia Pacific is anticipated to expand at the highest CAGR of 10.55% over the projection period.

Asia Pacific is expected to grow at the highest CAGR of 10.55% over the forecast period. It is due to increased public knowledge of stem cell's medical potential, as well as increased government spending in stem cell research and development. For many years, India has been at the forefront of medical advancements as one of the most popular foreign destinations for medical tourism. Furthermore, the development of novel treatments and procedures, as well as the higher success rate of stem cell treatment, are likely to drive expansion in the region's stem cell banking business.

The adult stem cells segment is expected to register the highest CAGR of 10.32% over the projected period in the stem cell banking market.

The adult stem cells segment is anticipated to grow at the highest CAGR of 10.32% in the stem cell banking market. The growing understanding of the variety and effectiveness of adult stem cell banking services is driving up demand. Adult stem cell preservation is being considered by patients, physicians, and researchers as a proactive strategy to future disease problems. This need promotes business competitiveness and innovation, resulting in enhanced storage systems and broader service offers.

Over the projected period, the sample preservation and storage segment is expected to register the highest CAGR of 10.73% in the stem cell banking market.

Over the forecasted period, the sample preservation and storage segment is anticipated to grow at the highest CAGR of 10.73% in the stem cell banking market. This vital service area includes cutting-edge cryopreservation processes, cutting-edge storage facilities, and stringent quality control systems. In this age of regenerative medicine, the efficiency of stem cell treatments is dependent on the quality and accessibility of preserved samples.

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Market Dynamics

Driver: A growing elderly population

An older population has a favourable impact on the market. This demographic shift is changing healthcare dynamics all around the world. As people age, they become more susceptible to degenerative diseases such as osteoarthritis, cardiovascular disease, and neurological disorders such as Alzheimer's and Parkinson's. Stem cells have immense promise for repairing damaged or ageing tissues, paving the way for new treatments and better quality of life for the elderly. This ageing population necessitates more modern healthcare treatments and represents a significant client base for stem cell banking services. Many people and families are aware of the option of keeping stem cells from themselves or loved ones, which can be taken from sources such as cord blood or adipose tissue. These stem cells can be used in future therapies to combat age-related health issues, offering comfort and hope.

Opportunity: Growing ethical issues over the use of embryonic stem cells

The market is being fueled by growing ethical concerns about the use of embryonic stem cells. Because embryonic stem cell research involves the killing of embryos, it has long been a subject of ethical debate, leading in moral and legislative constraints in a variety of domains. This has shifted the emphasis of stem cell research and therapeutic applications away from controversial sources and towards non-controversial sources such as adult stem cells and cord blood. As a result, it is becoming popular among individuals and institutions seeking the potential benefits of stem cell therapy without the ethical ambiguity of stem cell banking. Cord blood, in particular, has grown in prominence as a rich source of stem cells that is ethically sound. Families and healthcare practitioners recognise the value of keeping these cells as a form of biological insurance against future illnesses for the donor and potentially compatible family members.

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Some of the major players operating in the stem cell banking market are:

Cordlife Cryo-Save AG (A Group of Esperite) Stemcyte Smart Cells International Ltd. Cordvida CBR Systems, Inc. Lifecell Cryoviva India Cryo-Cell Viacord

Key Segments cover in the market:

By Product Type:

Human Embryonic Cells Adult Stem Cells IPS Cells

By Service Type:

Sample Analysis Sample Collection and Transportation Sample Preservation and Storage Sample Processing

By Region

North America (U.S., Canada, Mexico) Europe (Germany, France, U.K., Italy, Spain, Rest of Europe) Asia-Pacific (China, Japan, India, Rest of APAC) South America (Brazil and the Rest of South America) The Middle East and Africa (UAE, South Africa, Rest of MEA)

About the report:

The market is analyzed based on value (USD Billion). All the segments have been analyzed worldwide, regional, and country basis. The study includes the analysis of more than 30 countries for each part. The report analyzes driving factors, opportunities, restraints, and challenges for gaining critical insight into the market. The study includes porter's five forces model, attractiveness analysis, product analysis, supply, and demand analysis, competitor position grid analysis, distribution, and marketing channels analysis.

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Stem Cell Banking Market Size Revenue Hits $18.04 Billion by 2032 ... - GlobeNewswire

Scientists Created a Monkey With Two Different Sets of DNA – Smithsonian Magazine

The monkey "chimera" with two sets of DNA at three days old. Some body parts appear tinted green, because the researchers marked the transplanted cells with fluorescent dye to trace what parts they developed into. Cell / Cao et al.

Researchers have created a monkey with two different sets of DNAby injecting stem cells from one monkey embryo into another of the same species. This method has been used in rats and mice beforebut the recent feat marks the first time ever that it has been successful in another animal, including primates. Scientists say the breakthrough could help with medical research in the future.

This is a long-sought goal in the field, Zhen Liu of the Chinese Academy of Sciences (CAS) says in a statement. This work could help us to generate more precise monkey models for studying neurological diseases as well as for other biomedicine studies.

However, the monkey had to be euthanized after ten days due to breathing issues and hypothermia, which some scientists say highlights ethical concerns in this type of research, reports Nature News Carissa Wong.

In reference to the mythological, fire-breathing chimera that has a lions head, a goats body and a serpents tail, individuals that contain two or more different sets of DNA in their bodies are referred to as chimeric by scientists. Chimerism can occur naturally, as when one embryo in a set of fraternal twins dies in the womb and the other absorbs its cells. This has been documented in several species of birds, reptiles and mammals, including humans.

But chimerism can also occur artificially with an organ or bone marrow transplant. In this case, the researchers transplanted stem cells, which can develop into any kind of cell.

To create the monkey chimera, Liu and his colleagues removed stem cells from seven-day-old embryos of long-tailed macaques (Macaca fascicularis). They labeled these with green fluorescent protein so that any tissue the cells created in a chimeric monkey could be visually identified later. They then injected these cells into four- to five-day-old embryos of the same species and implanted them into 40 female macaques.

Of these surrogate mother monkeys, 12 became pregnant, and 6 gave birth to live young. The teams analysis showed that just one live-birth male and one miscarried male were substantially chimeric. In the live monkey, donor cells made up 67 percent of its tissues on average, but across the 26 different tissue types tested, that number ranged between 21 percent and 92 percent.

Scientists saw evidence of glowing green fluorescencethe mark of the donor cellsin the live monkeys fingertips and around its eyes. Percentages of donor cells in the miscarried fetus were lower. The team published its research this month in the journal Cell.

It is a very good and important paper, Jacob Hanna, a stem cell biologist and embryologist at the Weizmann Institute of Science in Israel who was not involved with the study, tells CNNs Katie Hunt. This study may contribute to easier and better making of mutant monkeys, just like biologists have been doing for years with mice. Of course, work with [nonhuman primates] is slower and much harder but is important.

Researchers have been creating chimeric mice since the 1960s to learn more about critical developmental processes, including how stem cells grow into more specialized cells. Theyve also used the mice as models to study diseases. But trying to understand humans by looking at rodents has its limitations.

Mice dont reproduce many aspects of human disease for their physiology being too different from ours, Liu tells CNN. In contrast, human and monkey are close evolutionary, so human diseases can be more faithfully modeled in monkeys.

In controversial research, scientists have previously created human-monkey chimeric embryos, though these only grew for 20 days before being destroyednot long enough to develop a brain or nervous system. Some scientists hope these techniques could be used to grow human organs inside other animals for transplantation, per Nature News. But such efforts involving animalsespecially once human cells are addedcan quickly pose ethical quandaries.

All animal research warrants careful consideration, but this is particularly important for all non-human primate research, stem cell researcher Megan Munsie, of the University of Melbourne and Murdoch Childrens Research Institute, tells Peter de Kruijff of the Australian Broadcasting Corporation (ABC).

Munsie notes to the publication that, of all 74 chimeric monkey embryos transferred into surrogate mothers in the recent study, only one living macaque produced the desired resultsand it had to be euthanized. Future efforts should focus on improving embryo viability to avoid the high abortion rate and associated distress and waste, she adds.

Additionally, long-tailed macaques, while commonly used as lab monkeys, were listed as endangered by the International Union for Conservation of Nature last year. Munsie suggests limiting research to animals that are not endangered, per the ABC. The authors, however, say this research could help with conservation efforts.

Monkey chimeras also have potential enormous value for species conservation if they could be achieved between two types of nonhuman primate species, one of which is endangered, co-author Miguel Esteban, of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, and a researcher with BGI-Research Hangzhou, tells CNN. If there is contribution of the donor cells from the endangered species to the germ line, one could envisage that, through breeding, animals of these species could be produced.

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Scientists Created a Monkey With Two Different Sets of DNA - Smithsonian Magazine

Metastases and treatment-resistant lineages in patient-derived … – Nature.com

Selection of patients and 2DOs

Eight patients with distant metastases or recurrences that could be evaluated using computed tomography were selected. All patients underwent baseline imaging within 4 weeks before anticancer drug administration. The tumor volume and reduction rate were measured according to RECIST guidelines42. 2DOs were established from primary CRC specimens and cultured according to a previous report20 and stocked at our laboratory cell bank. Briefly, CRC tissue from resected specimens was minced into 1-mm pieces and dissociated with 1mg/mL of collagenase (C6885; Sigma-Aldrich, St. Louis, MO, USA). Filtered cell pellets between 20m and 200m were seeded in plates coated with iMatrix-511 (Takara Bio Inc., Kusatsu, Japan) and cultured in medium containing 10ng/mL of basic fibroblast growth factor (ThermoFisher Scientific, Waltham, MA, USA) and 2ng/mL of transforming growth factor beta (R&D Systems Inc., Minneapolis, MN, USA) to maintain heterogeneous primary culture cells. Sixteen 2DOs with stable culture and drug sensitivity on testing, including eight 2DOs from patients with distant metastases or recurrences, were selected for further analysis.

The human colorectal tumor cell lines, HCT116, gifted by Dr. Bert Vongelstein (Johns Hopkins University, Baltimore, MD, USA), and HT29 (EC91072201, ECACC), were cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific), 1% GlutaMAXI (ThermoFisher Scientific), and 1% penicillin/streptomycin/amphotericin B (Wako Pure Chemical Industries, Osaka, Japan). Cells were incubated at 37C in a humidified atmosphere containing 5% CO2. Cells were harvested using 0.25% Trypsin-EDTA (ThermoFisher Scientific) for further analysis.

Cellartis human iPS cell line 12 (ChiPSC12) cells (Takara Bio) were cultured in the Cellartis DEF-CS 500 Culture System (Takara Bio). Cells were incubated at 37C in a humidified atmosphere containing 5% CO2. Cells were harvested using Accutase (Innovative Cell Technologies, Inc., San Diego, CA) for further analysis.

The expression of proteins in cells was determined using flow cytometry. Cultured cells were dissociated with Accutase (Nacalai Tesque Inc., Kyoto, Japan). CTCs were isolated from clinical blood samples using OncoQuick (Greiner BioOne, Frickenhausen, Germany) according to the manufacturers protocol. Cells were stained with antibodies targeting EpCAM, CD133, CD44, CD41, CD45, and LGR5 (Supplementary TableS2). For detecting POU5F1, a True-Nuclear Transcription Factor Buffer Set (424401; BioLegend) was used. After staining cell surface proteins, cells were fixed and stained with antibodies for POU5F1, according to the manufacturers protocol. Relative fluorescent intensities were measured with an SH800 cell sorter (SONY, Tokyo, Japan) and cell morphology and staining locations were also measured with a FlowSight imaging flow cytometer (Merck-Millipore, Darmstadt, Germany). 7-AAD (Miltenyi Biotec, San Diego, CA, USA) was used to analyze living cells. A dimensionality reduction step in two dimensions was performed using t-distributed stochastic neighbor embedding (t-SNE) to visualize high-dimensional data of stem cell marker expression. Data were analyzed using FlowJo 10.2 software (FlowJo LLC, Ashland, OR, USA).

Anticancer drug sensitivity was examined in sixteen 2DOs within 510 passages. Drugs and their concentrations in clinical drug assays are listed in Supplementary TableS3. The number of viable cells in each well was measured using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) before drug administration and 96h after drug administration. Cell proliferation in DMSO and distilled water, which were used to dilute each drug, were used as controls. The ratio of the number of living cells after administering the drug to the control is shown. Three independent experiments were performed and the average is shown. The formula used for calculation was as follows: 100Cont. 0h cell num.Drug 96h cell num./{(Cont. 96h cell num.Cont. 0h cell num.) Drug 0h cell num.}

Regarding the sensitivity of each anticancer drug, a dimensionality reduction step in two dimensions was performed using t-SNE to visualize high-dimensional data for 21 drugs in a low-dimensional space. The statistical analyses were performed using R 3.6.3 (R Core Team, 2018), with the data.table (v1.12.8; Dowle & Srinivasan43), t-SNE (Krijthe44), and ggplot2 (Wickham45) packages.

Total RNA was extracted using an RNA Purification Kit (Qiagen, Hilden, Germany). TruSeq Stranded mRNA Library Prep (Illumina, San Diego, CA, USA) was used to prepare RNA-seq libraries from the total RNA (1g). Multiplexed libraries were sequenced on an Illumina NextSeq with single-end 75-bp sequencing. RNA-seq data were mapped to the hg38 genome release using the bioinformatic pipeline of the Illumina Base Space Sequence Hub and the Subio software platform (Subio, Inc., Kagoshima, Japan).

The vector, PL-SIN-Oct4-EGFP, kindly provided by James Ellis (Addgene plasmid #21319; http://n2t.net/addgene:21319)22, was used to establish cells expressing EGFP under the OCT4 (POU5F1) promoter. The vector was transfected into 2DOs and cell lines using Lentiviral High Titer Packaging Mix with pLVSIN (Takara Bio). EGFP-positive cells were purified by sorting using a SH800 cell sorter (SONY) at least twice. POU5F1 expression was confirmed by polymerase chain reaction (PCR).

Total RNA was isolated using an RNA Purification Kit (Qiagen). Quantitative assessment was performed by real-time PCR using 100nM universal probe libraries, 0.1 FASTStart TaqMan Probe Master (Roche Diagnostics, Basel, Switzerland) for designed primers, iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) for commercially available primers, 100nM primers, and 10ng cDNA for cDNA amplification of target genes. Primers are listed in Supplementary TableS4. PCR was performed with 20L of the master mix in each well of a 96-well plate, and signals were detected with the CFX Connect Real-Time PCR Detection System (Bio-Rad). The thermocycler was programmed for one cycle at 95C for 10min, followed by 40 cycles at 94C for 10s, 60 C for 20s, and 72 C for 1s. cDNAs from NTERA-2 cells were used as positive controls.

A subcutaneous model was established to investigate the ability to differentiate from a single sorted cell. A single sorted cell was cultured in a dish for expansion using the 2DO culture methods described above. Accutane-dissociated cells (1106 cells) suspended in Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were subcutaneously transplanted into the dorsal flanks of 7-week-old, non-obese diabetic/severe combined immunodeficient mice (CLEA, Tokyo, Japan). The average weight was 27g at the start of the experiments. The mice were sacrificed when the tumors reached a diameter of 10mm. For the liver metastasis model, live cells (1106 cells) were sorted by 7-AAD (Miltenyi Biotec) according to EGFP expression using a SH800 cell sorter (SONY) and injected into the spleen. Liver metastasis was assessed every 4 weeks. Mice were sacrificed 8 weeks after injection for the assessment of liver metastases in the POU5F1 expression metastatic ability experiment and 10 weeks after injection in the XAV939 experiment.

Xenograft tumors were fixed in formalin, processed through a series of graded concentrations of ethanol, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (H&E). Three-dimensional (3D)-formed 2DOs cultured on a NanoCulture plate were collected and centrifuged at 400g for 5min at room temperature. The pellet was consolidated using iPGell (GenoStaff Co., Ltd., Tokyo, Japan) and fixed in formalin. The pellet was processed through a series of graded concentrations of ethanol, embedded in paraffin, sectioned, and stained with H&E.

Xenograft tumors were also fixed in 10% buffered formalin and embedded in paraffin blocks. For cultured 2DOs, 3D-formed 2DOs cultured on an Ultra-Low Attachment Multiple Well Plate (Corning, NY, USA) were collected and centrifuged at 400g for 5min at room temperature. They were embedded in paraffin blocks using iPGell (GenoStaff). A 3-m section was obtained from each block. Sections were deparaffinized, and slides were boiled for 15min. Expressions of CD44, CK20, MUC2, and chromogranin A were quantified using antibodies (Supplementary TableS5). The slides were incubated with a primary antibody for 60min at room temperature and then incubated with a secondary antibody for 30min at room temperature. Slides were mounted in Prolong Gold with DAPI (Invitrogen, Waltham, MA, USA). Mucus production ability was assessed via Alcian blue staining (pH 2.5).

Cultured cells were fixed with 4% formaldehyde and blocked. They were incubated with primary antibodies (Supplementary TableS6) overnight at 4C. Cells were incubated with secondary antibodies for 90min. Slides were mounted in Prolong Gold with DAPI (ThermoFisher Scientific) overnight.

The vector, pLV[Exp]-Neo-CMV>DsRed_Express2, was constructed by VectorBuilder, Inc. (Chicago, IL, USA) (Supplementary Fig.S27). This vector was transfected into 2DOs and iPS cells using Lentiviral High Titer Packaging Mix with pLVSIN (Takara Bio). DsRed_Express2-positive cells were selected by antibiotic selection using G418 (10131035; ThermoFisher Scientific) and sorted twice by an SH800 cell sorter (SONY). All cells expressing DsRed-Express2 were detected by an SH800 cell sorter (SONY).

The vector, PL-SIN-Oct4-EGFP, kindly provided by James Ellis (Addgene plasmid #21319)22, and the vector, pMSCV-F-del Casp9.IRES.GFP, kindly provided by David Spencer (Addgene plasmid # 15567)46, were used to establish cells expressing EGFP under the OCT4 (POU5F1) promoter with inducible caspase 9. Sequence-encoding caspase 9 was digested with restriction enzymes, XhoI (R0146S; New England Biolabs, Beverly, MA, USA) and EcoRI-HF (R3101S; New England Biolabs). The DNA fragment of caspase 9 was extracted from E-Gel CloneWel 0.8% (G6500ST; ThermoFisher Scientific) using the E-Gel Power Snap Electrophoresis System (ThermoFisher Scientific) (Supplementary Fig.S28). The fragment was amplified using CloneAmp HiFi PCR Premix (Z9298N; Takara Bio) with designed primers (FW_gaattctgcagtcgatcgagggagtgcaggtgg, RV_ccgcggtaccgtcgacttagtcgagtgcgtagtc). The vector, PL-SIN-Oct4-EGFP, was linearized by a restriction enzyme, SalI-HF (R3138S; New England Biolabs). The amplified fragments and linearized vector were used for the cloning reaction by the In-Fusion HD Cloning Kit (Z9648N; Takara Bio). The transformation procedure was performed using Competent High E. Coli DH5 (TYB-DNA903; Toyobo, Osaka, Japan), and the plasmid was extracted using the Qiagen Plasmid Plus Midi Kit (12945; Qiagen). The nucleotide sequence of the vector was confirmed by Sanger sequencing, performed by GENEWIZ Japan Corp. (Kawaguchi, Japan). Primer extension sequencing was performed using Applied Biosystems BigDye version 3.1, and the reactions were then run on an Applied Biosystem 3730xl DNA Analyzer. The constructed vector was transfected into two 2DOs (603iCC and 25DiCC) using Lentiviral High Titer Packaging Mix with pLVSIN (Takara Bio). EGFP-positive cells were cloned by single-cell sorting using an SH800 cell sorter (SONY). POU5F1 expression was confirmed by PCR, and a decrease in the number of EGFP-positive cells was confirmed by the administration of B/B Homodimerizer (Z5059N; Takara Bio). The mean provirus copy number was 6.05 (1.16, n=6), as measured using the Let-X Provirus Quantitation Kit (Z1239N; Takara Bio).

603iCC-transfected POU5F1-EGFP cells with inducible caspase 9 (4.5104/well) were seeded, and 5M B/B Homodimerizer (Takara Bio) was administered for three days. Four days after the dimerizer was removed, live cells were sorted using an SH800 cell sorter (SONY) as day 7 cells. For cells not treated with a dimerizer, live cells were also sorted as day 0 cells. Single-cell library preparation was performed following the manufacturers instructions for the Chromium Next GEM Single Cell 3 Reagent Kit (v3.1) (10x Genomics, Pleasanton, CA, USA), and the libraries were sequenced on a HiSeq X sequencer (Illumina). To generate a data matrix, the Cell Ranger pipeline (v4.0.0) was applied, and raw reads were aligned to the human reference genome (GRCh 38) using the STAR aligner. For GFP transcript mapping, the GFP sequence (XM_013393261) was added to the reference fastq and gtf files. Data were deposited in Gene Expression Omnibus under the accession number GSE169220.

Seurat (version 3.2.0)47 was used for quality control and downstream analysis. Poor-quality cells were filtered out using the following parameters: nFeature_RNA 2009000 and percent.mt <10. A total of 6942 cells (control: 3342 cells and day 7: 3602 cells), which passed the quality control, were finally used for further analysis. Mitochondrial genes were filtered by mt.percent (<10). UMAP visualization was used for dimensionality reduction analysis with the following parameters: resolution, 0.5; and perplexity, 20. Marker genes discriminating the different clusters were identified using the FindAllMarkers function (min.pct=0.25 and log[fold-change] >0.25). Pathway enrichment analysis was performed using Enrichr48 (https://maayanlab.cloud/Enrichr/). To construct a single-cell pseudotime trajectory, the Monocle3 (v0.2.2) algorithm was applied (https://cole-trapnell-lab.github.io/monocle3/). After converting the Seurat object using the as.cell_data_set function, the root node was assigned to cluster 4, and the orderCells function was used to assign cells a pseudotime value. To subdivide cells based on their branch in the trajectory, the choose_graph_segments function was applied, and cluster 6 was chosen as an ending node.

Western blot analysis was performed to examine proteins associated with the Wnt/-catenin signaling pathway. Cells were lysed in 50mM TrisHCl (pH 7.6), 1% Nonidet P-40, 150mM sodium chloride, and 0.1mM zinc acetate in the presence of protease inhibitors. Protein concentration was determined by the Lowry method (Bio-Rad), and 20g of each sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was transferred electrophoretically onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with blocking buffer for 1h and then incubated overnight at 4 C with primary antibodies against -catenin (1:1000, 8480, Cell Signaling Technology), Wnt-3a (1:5000, GTX128101, Gene Tex, CA, USA), and HistoneH3 (1:2000, 4499S, Cell Signaling Technology). After a 2-h incubation with the secondary antibody, horseradish peroxidase-conjugated rabbit antibody (1:400, 7074S, Santa Cruz Biotechnology Inc., Dallas, TX, USA), protein bands were visualized using an ECL detection kit (ThermoFisher Scientific) according to the manufacturers instructions.

DNA samples were treated with sodium bisulfite using a bisulfite conversion kit (Zymo Research EZ DNA methylation Kit). After treatment, unmethylated cytosines convert to uracil, while methylated cytosines remain unchanged. Bisulfite-converted DNA samples were analyzed using the Infinium MethylationEPIC BeadChip Kit (Illumina). Bisulfite-converted DNA samples were denatured and neutralized by alkali. The denatured samples were then amplified by whole-genome amplification (37C overnight). Amplified DNA samples were enzymatically fragmented for 1h at 37C in a microsample incubator. 2-Propanol was added to the fragmented DNA samples and precipitated by centrifugation. Precipitated DNA samples were resuspended with hybridization buffer and incubated for 1h at 48C in a hybridization oven. Fragmented and resuspended DNA samples were denatured for 20min at 95C in a microsample incubator. Denatured DNA samples were dispensed onto BeadChips using a TECAN System. The BeadChips were incubated overnight at 48 C in the hybridization oven to hybridize the samples onto the BeadChips. After hybridization, seals were removed from the hybridized BeadChips. Next, unhybridized fragment DNAs were washed away. Labeled nucleotides were added to the washed BeadChips to extend primers which hybridized to the DNA. BeadChips were stained, then coated for protection, and dried. Dried BeadChips were scanned with the iSCAN System. Illumina GenomeStudio software (V2011.1) loaded the signal intensity files of BeadChips, and beta values were decided via normalization and background subtraction. Next, a comparative analysis was executed based on the Illumina Custom Model algorithm, and difference scores for all probes were computed. The markers with signal intensities adequate to distinguish between the signal and background noise were used in subsequent analysis. The markers with high scores (highly methylated and highly unmethylated compared to the reference sample) were extracted, and clustering analysis was conducted.

The NANOG binding consensus sequence is generally known to be 5TAAT[GT][GT]3 or 5[CG][GA][CG]C[GC]ATTAN[GC]3. Therefore, in the sequence of focus, the CGCCCAGTGTC part is quite similar to the binding sequence. We used Protein Data Bank data, including 4RBO, to predict binding conformations to the NANOG protein with the wild-type sequences or methylated sequence with our original method49. A sufficient amount of water molecules was placed around the complex structures, and thermodynamical sampling was performed under a periodic boundary condition. After stabilizing the complex structure by energy minimization calculations, some molecular dynamics simulations were performed at ~37C (310K) to capture the molecular behavior under the biological environment. After a sufficient thermal equilibration process, the molecular vibrations of the bonding configurations were sampled. All these calculations were performed using the AMBER package. The distributions of the interaction energy between DNA and NANOG protein were calculated by extracting 2000 conformations of complex structures from the trajectory with the abovementioned molecular dynamics simulations. Each binding energy was calculated using Gaussian program packages50 with the AMBER99 Force field level51.

603iCC cells (1104 per well) were seeded into 96-well plates and incubated for 48h. After incubation, cells were exposed to different concentrations of XAV939 (BD248591; BLD Phamatech Ltd., Shanghao, China) for 96h. The percentage of viable cells was determined using a cell counting kit solution (CCK-8; Dojindo Molecular Technologies) according to the manufacturers protocol.

Prior to cancer cell seeding, plates were coated. iPS cell-coated plates were seeded into 12-well plates (2105 iPS cells/well) 2 days prior to seeding. iPS cells were tagged with DsRed-Express by the aforementioned methods. Laminin coatings were prepared using iMatrix-511 (T304, Takara Bio) according to the manufacturers protocol. Sorted POU5F1-positive cells (2105/ well) were seeded on these plates. Medium was prepared with XAV939 (10M) for the XAV939 group and DMSO (0.3%) for the control group. All medium exchanges were performed every other day, and cells in the collected supernatant were analyzed by an SH800 cell sorter (SONY). Cells not expressing DsRED-Express2 were counted as cancer cells.

Stained specimens were analyzed using ImageJ software52. Five independent images were collected for each sample and the areas of protein expression in the samples were measured. The value was normalized by dividing by the number of cells stained with DAPI.

As an evaluation of XAV939, sorted POU5F1-positive cells were directly injected into the spleen of mice (1106 cells). After recovering from anesthesia, mice were randomly allocated to the control (0.3% DMSO that is the final concentration of DMSO in XAV939 group) or XAV939 group (100g/injection/mouse). XAV939 (CS-0494, ChemScene, Monmouth Junction, NJ, USA) was administered by intraperitoneal injection at 1mg/mL (injection volume, 100L) every day for 8 weeks, followed by 2 weeks of observation. Ten weeks after injection, mice were sacrificed for the assessment of metastases. Mouse body weight was measured twice per week, and no weight gain or loss greater than 5% was observed.

The Osaka University Review Board, the OICI Review Board, approved this study, and written informed consent for the study was obtained from all participants according to the ethics guidelines. All ethical regulations relevant to human research participants were followed. The OICI Animal Research Committee approved this study, and we have complied with all relevant ethical regulations for animal use. All experimental protocols were in accordance with the guidelines of the Osaka University, the OICI, and Declaration of Helsinki.

Continuous variables are expressed as the mean with standard error of the mean. The significance of the difference between the two groups was analyzed using the x2 test and Wilcoxons signed rank-sum test. All data were analyzed using JMP software (SAS Institute), R 3.6.3, and Prism 8 (GraphPad Software, San Diego, CA, USA). Results were considered statistically significant at P<0.05.

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