Category Archives: Embryonic Stem Cells

Human stem-cell-based therapy for Parkinson’s disease proven safe PET – BioNews

A small clinical trial involving 12 patients with Parkinson's disease has reported no safety concerns with a newly developed human stem-cell-based therapy.

The therapy called TED A9 was delivered as a cell transplant injected directly into the brain of the participants as part of a Phase 1/2a clinical trial, which is principally concerned with assessing safety and dosing requirements.

The drug's developer, S.Biomedics, in Seoul, South Korea, claimed in a press release: 'According to Professor Jin-Woo Chang, [the principal investigator of the transplant conducted at Severance Hospital, Seoul,] none of the 12 Parkinson's disease participants had any side effects, complications, or unusual adverse reactions following the transplantation of TED-A9'.

The trial participants were aged between 50 and 75 years old, had been diagnosed with Parkinson's disease for more than five years, and had already motor complications such as freezing of gait or dyskinesia.

To ensure and monitor the safety of the treatment, an initial three patients were injected with a low dose (3.15 million cells) and monitored for three months, before another three patients were treated at high dose (6.3 million cells) and also monitored for three months.

No side effects, complications, or unusual adverse reactions were seen in either group during the three-month assessment period. Therefore, the clinical trial continued by adding three further patients to each of the low-dose and high-dose groups. Again, no safety concerns were seen.

Parkinson's symptoms are caused by the progressive loss of neurons that produce dopamine, a major chemical messenger in the brain. The TED-A9 therapy contains dopaminergic progenitor (precursor) cells, which had themselves been derived in the lab from embryonic stem cells.

The drug developers at S.Biomedics hope that the dopaminergic precursor cells in TED-A9 will treat Parkinson's disease by replacing the mature dopamine-producing nerve cells that are lost in patients.

Professor Dong-Wook Kim, a neurosurgeon and the principal developer of TED-A9, said: 'We have developed a fundamental therapeutic mechanism that directly replaces dopaminergic neurons lost in patients with Parkinson's disease. TED-A9 could represent a fundamental treatment that surpasses current therapies, which only temporarily alleviate the symptoms of Parkinson's disease,'.

The trial is expected to continue until February 2026, allowing safety of the therapy to be monitored for a total of five years. As part of the study, exploratory efficacy will also be examined for two years using clinical measures of motor symptoms and a patient questionnaire of daily life quality.

More Information is available at ClinicalTrials.gov.

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Human stem-cell-based therapy for Parkinson's disease proven safe PET - BioNews

Unlocking the Secrets of Aging: Researchers Reveal Key to Intestinal Balance – SciTechDaily

University of Helsinki researchers discovered that the capacity of intestinal stem cells to maintain cellular balance in the gut diminishes with age, and identified a new mechanism linking nutrient adaptation of these stem cells to aging. This insight could lead to methods for preserving gut function in the elderly.

The ability of intestinal stem cells to preserve the cellular equilibrium in the gut diminishes with age. Scientists at the University of Helsinki have identified a novel interaction between the adaptation of intestinal stem cells to nutrients and the aging process. The finding may make a difference when seeking ways to maintain the functional capacity of the aging gut.

The cellular balance of the intestine is carefully regulated, and it is influenced, among other things, by nutrition: ample nutrition increases the total number of cells in the gut, whereas fasting decreases their number. The relative number of different types of cells also changes according to nutrient status.

The questions of how the nutrition status of the gut controls stem cell division and differentiation, and how the nutrient adaptation of stem cells changes as during aging have not been comprehensively answered. Nutrient adaptation refers to the way in which nutrients guide cell function.

On the left: Model organism fruit fly (Drosophila melanogaster), gastrointestinal tract highlighted in green. On the right: Microscope images of the fruit fly intestine where cell nuclei are stained (cyan). The intestine on the top is from well-fed animal, and the intestine below from an animal kept on a restricted diet. Credit: Jaakko Mattila

Researchers at the University of Helsinki identified a new regulatory mechanism that directs the differentiation of intestinal stem cells under a changing nutrient conditions. Cell signaling activated by nutrients increases the size of stem cells in the fruit fly intestine. The size of the stem cells, in turn, controls the cell type into which the stem cells differentiate. For stem cell function, flexible regulation of their size is essential.

In other words, the size of the cells dynamically increases or decreases, depending on the dietary conditions. Such flexibility enables stem cells to differentiate in accordance with the prevailing nutrient status. By utilizing intestine-wide cell imaging, the researchers found that the nutrient adaptation of stem cell size and the resulting differentiation vary in different regions of the gut.

Our observations demonstrate that the regulation of intestinal stem cells is much more region-specific than previously understood. This may be relevant to, for example, how we think about the pathogenetic mechanisms of intestinal diseases, says Jaakko Mattila, the corresponding author of the research article from the Faculty of Biological and Environmental Sciences, University of Helsinki.

The researchers also observed that the ability of intestinal stem cells to react to a changing nutrient status is greatly reduced in older animals. They also found that, in older animals, stem cells are in a state where they are constantly large in size, which restricts their ability to differentiate. With aging, flexible regulation of stem cell size was markedly better preserved in animals that had been kept under a diet regime that is known as intermittent fasting. In the past, intermittent fasting has been shown to prolong the lifespan of animals, and the results now obtained indicate that the improved preservation of stem cell function may underlie this prolongation.

According to the researchers, the mechanisms associated with the functioning, nutrient adaptation, and aging of human and fruit fly stem cells are fairly similar.

We believe that these findings have a broader significance towards understanding how to slow down the loss of tissue function caused by aging by controlling the nutrient adaptation of stem cells. However, more information is needed on the effect of the mechanism on human intestinal stem cells. Our work on the nutrient adaptation of stem cells continues, says Professor Ville Hietakangas from the Faculty of Biological and Environmental Sciences and the Institute of Biotechnology, University of Helsinki.

Reference: Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation by Jaakko Mattila, Arto Viitanen, Gaia Fabris, Tetiana Strutynska, Jerome Korzelius and Ville Hietakangas, 9 February 2024, Science Advances. DOI: 10.1126/sciadv.adi2671

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Unlocking the Secrets of Aging: Researchers Reveal Key to Intestinal Balance - SciTechDaily

Stem Cells. Let’s get things straight about what they are, and what they are not. Ethically and legally Sonoran News – Sonoran News

Once again, I receive so many emails regarding stem cells, and where they come from. This is the biggest topic of questions I receive each week. The first thing Id like to say is that in my office, Accurate Care Medical Wellness Center, only ethical and legal sources of stem cells are used. We also use cells and protocols that are the most effective for each condition a patient may have. I personally go to three stem cell conferences a year to stay current with all of the products and protocols that are available.

It seems that people have been told about what used to be done back when stem cell therapy started. Very little information is available to the public that explains the recent technology and sources of stem cells and their ability to restore function to otherwise dysfunctional systems of the body to promote healing. I will do my best to shed light on this subject, while clarifying what they are and what they are not.

Lets start with what stem cells we do not use in my office. Embryonic stem cells are derived from embryos that are typically created through in vitro fertilization (IVF) procedures for reproductive purposes. These unused embryos are typically donated for research purposes with the informed consent of the donors. In some cases, aborted embryos may be used. In 2001, then-President George W. Bush announced a policy limiting federal funding for research involving embryonic stem cells. This policy allowed federal funding only for research on embryonic stem cell lines that had already been established before August 9, 2001. In 2009, President Barack Obama issued an executive order that lifted the restrictions on federal funding for embryonic stem cell research. This allowed for the funding of research on new embryonic stem cell lines. We do not use embryonic stem cells for treatments in my office, as we do not use products that are illegal, unethical or controversial.

Amniotic stem cells are derived from the amniotic fluid and amniotic membrane surrounding the fetus during pregnancy, generally obtained through procedures like amniocentesis, the umbilical cord (in stem cell therapy for labs), or during a cesarean section childbirth. Unlike embryonic stem cells, which are derived from embryos, amniotic stem cells are obtained without harming the fetus and are ethically uncontroversial. We use MSCs or Mesenchymal Stem Cells, and extra cellular cells or exosomes in my office.

We use stem cells, now known as HCT or Human Cellular Tissue Therapy. This term is now used, as not all products used for treatments today contain stem cells, Some are extracellular products. Extracellular vesicles (EVs)derived from mesenchymal stem cells, (MSCs) play a critical role in the development of immune regulation and regeneration. These mimic the effects of stem cells and perform powerful functions. In my office, each patient, case and condition is unique, therefore different products and protocols are used for each and every patient. As work continues, researchers are actively developing engineered EVs that are even more effective.

Many patients tell me that theyve previously received the following types of stem cells when they received stem cell therapy that did not work prior to coming to my office.

Two of the popular forms of stem cell therapy theyve received are adipose (fat) and bone marrow derived cells. These two sources of stem cells can work well for younger patients. The challenge that arises with older patients, especially those over the age of 60, is that these cells come from an older body.

Many of the regenerative properties of the cells have been used up and overworked over the years. This leaves the patient with compromised cells that are not going to regrow the tissue in question to the level and expectations the patient and doctor are willing to see. The two forms of stem cell extraction from the patient are invasive, and can be quite painful, especially those from bone marrow. Using stem cells, and extracellular cells from the donated umbilical cord of live birth of a baby is much less invasive for the donor (baby) and the recipient (patient).

For any questions regarding regenerative medicine, and whether it may help you, please call my office for a complimentary consultation. I offer special discounts for my readers as well. We frequently offer evening lectures in my office to learn more about regenerative medicine. If you would like to be added to our list for future events, please call my office.

For questions regarding any of my articles, please email me at [emailprotected] Leisa-Marie Grgula. DC Chiropractic Physician Accurate Care Medical Wellness Center 18261 N. Pima Rd. Ste. #115 Scottsdale, AZ 85255 480-584-3955

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Stem Cells. Let's get things straight about what they are, and what they are not. Ethically and legally Sonoran News - Sonoran News

Distinct pathways drive anterior hypoblast specification in the implanting human embryo – Nature.com

Ethics statement

Human embryo work was regulated by the Human Fertility and Embryology Authority under licence R0193. Approval was obtained from the Human Biology Research Ethics Committee at the University of Cambridge (reference HBREC.2021.26). All work is compliant with the 2021 International Society for Stem Cell Research (ISSCR) guidelines. Patients undergoing IVF at CARE Fertility, Bourn Hall Fertility Clinic, Herts & Essex Fertility Clinic, and Kings Fertility were given the option of continued storage, disposal or donation of embryos to research (including research project specific information) or training at the end of their treatment. Patients were offered counselling, received no financial benefit and could withdraw their participation at any time until the embryo had been used for research. Research consent for donated embryos was obtained from both gamete providers. Embryos were not cultured beyond day 14 post-fertilization or the appearance of the primitive streak. Human stem cell work was approved by the UK Stem Cell Bank Steering Committee (under approval SCSC21-38) and adheres to the regulations of the UK Code of Practice for the Use of Human Stem Cell Lines. Mice were kept in an animal house in individually ventilated housing on 12:12h lightdark cycle with ad libitum access to food and water. Ambient temperature was maintained at 2122C and humidity at 50%. Experiments with mice are regulated by the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and carried out following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. Experiments were approved by the Home Office under licences 70/8864 and PP3370287. CD1 wild-type males aged 645weeks and CD1 wild-type females aged 618weeks were used for this study. Animals were inspected daily, and those showing health concerns were culled by cervical dislocation.

Raw fastq files from human datasets26,27,36,45, cynomolgus monkey datasets28,35 and mouse datasets68,69,70,71 were obtained from public repositories with wget. All human datasets were aligned to the GRCh38 reference using kb-pythons kb ref function to generate a reference. For cynomolgus monkey, National Center for Biotechnology Information (NCBI) genome build 5.0 transcriptome fasta files were adjusted to Ensembl style and used in kb ref to generate a custom index. For the mouse, GRCm39 reference was used with kb ref to generate a custom index. All datasets were re-aligned using either kb-python or kallisto72,73, after data handling as below. Human datasets: 10x v2 data from Mol et al. were processed as previously described10. For Zhou et al.27, read1 files were trimmed using cutadapt74 for the reported adapter sequence. Trimmed reads were then aligned using the kb-python kb count function with custom specifications (-x 1,0,8:1,8,16:0,0,0) and the custom barcode whitelist available. Each pair of fastqs was processed individually into barcodegene matrices and concatenated. For Xiang et al.26, a batch file was generated with cell ID, read1 and read2 for each fastq pair listed. Kallistos pseudo quant command was then used to generate a cell IDgene matrix. For Blakely et al.36, reads were aligned using kallisto pseudo quant. For Petropoulos et al.45, single-end reads were processed with kallisto pseudo quant with a pre-made batch file as above with 43 base pair read length specified. Cynomolgus datasets: for Ma et al.28, read1 fastqs were trimmed using cutadapt for TSO and polyA tail as described in the original publication. Next, kb pythons kb count function was used with custom specifications (x 1,0,8:1,8,16:0,0,0). For Yang et al.35, reads were aligned using kb pythons kb count command with 10xv3 technology specified. For Nakamura et al.21, available count tables were used given the use of SOLiD sequencer limiting re-alignment program options. Mouse datasets: for Mohammed et al.70, kallisto pseudo quant with a generated batch file was used to generate a cell IDgene matrix. For Deng et al.69 and Cheng et al.68, single-end reads were aligned with kallisto pseudo quant. Finally, for Pijuan-Sala et al.71, each sample set of 33 fastq files was aligned with kb count, with 10xv1 technology specified. The resulting set of barcodegene matrices was then concatenated for downstream analysis.

Following re-alignment, any datasets not generated using unique molecular identifier counts were normalized using quminorm75. First, matrices were converted to transcripts per kilobase million (TPM), and then the TPM matrix ran through quminorm with a shape parameter up to a maximum of 2 that did not create not available/applicable (NA) values in the matrix. Then, each individual dataset was made into a Seurat object76. Each individual dataset was then merged into a species-specific Seurat object, with SCT batch correction applied across datasets. Clusters were identified on the basis of canonical marker expression. To perform module scoring, gene lists were obtained from rWikiPathways77. For the monkey and mouse, gene symbols were converted to human homologues using bioMart78. Seurats AddModuleScore function was used with WikiPathway gene lists of interests as input. For CellPhoneDB analysis41, human data were split on the basis of stage, and subset matrix and metadata for cell type were output as txt files. CellPhoneDB was then run with respective files and counts-data set to gene_name. Data visualization was performed using Seurats DimPlot, FeaturePlot and VlnPlot functions, Scillus (https://scillus.netlify.app) Plot_Measure function, pheatmap and CellPhoneDBs dotplot function.

The scripts used for analyses are available at ref. 79.

Human embryos were thawed and cultured as described previously10,24. Briefly, cryopreserved human blastocysts (day 5 or 6) were thawed using the Kitazato thaw kit (VT8202-2, Hunter Scientific) according to the manufacturers instructions. The day before thawing, TS solution was placed at 37C overnight. The next day, IVF straws were submerged in 1ml pre-warmed TS for 1min. Embryos were then transferred to DS for 3min, WS1 for 5min and WS2 for 1min. These steps were performed in reproplates (REPROPLATE, Hunter Scientific) using a STRIPPER micropipette (Origio). Embryos were incubated at 37C and 5% CO2 in normoxia and in pre-equilibrated human IVC1 supplemented with 50ngml1 insulin growth factor-1 (IGF1) (78078, STEMCELL Technologies) under mineral oil for 14h to allow for recovery. Following thaw, blastocysts were briefly treated with acidic Tyrodes solution (T1788, Sigma) to remove the zona pellucida and placed in pre-equilibrated human IVC1 in eight-well -slide tissue culture plates (80826, Ibidi) in approximately 400l volume per embryo per well. Half medium changes were done every 24h. For small-molecule experiments, human IVC1 was supplemented with either 2M A83-01 (72022, STEMCELL Technologies)80,81, 25ngml1 Activin-A (Qk001, QKINE)82,83,84, 200nM LDN (S2618, SelleckChem)85,86, 50ngml1 BMP6 (SRP3017, Sigma Aldrich)85,86, 20M DAPT (72082, STEMCELL Technologies)87,88,89,90, 10M Compound-E (ab142164, Abcam)91,92,93, 20M MK-0752 (S2660, Selleck Chemicals)94,95,96 or dimethyl sulfoxide (DMSO) for 48h. In all cases, these concentrations fall within a range of those used for either vertebrate embryos or complex human ES cell-derived models of the embryo. Within these ranges, a low-to-intermediate concentration was selected to avoid non-specific cytotoxic effects while still considering the higher concentration needed for embryo permeation compared with minimal 2D cell culture to achieve inhibitor action. Further, all small molecules and proteins were tested on human ES cells to validate the efficacy and test for cytotoxicity. For analysis, embryos were fixed in 4% paraformaldehyde for 20min at room temperature for downstream analysis.

Pregnant, time-staged mice were culled by cervical dislocation, and uteri were dissected and placed in M2 medium (pre-warmed if embryos were for in vitro culture, ice cold if for fixing). E3.5 blastocysts were flushed out of uteri of pregnant females and either fixed for immunofluorescence analysis or transferred to acidic Tyrodes solution for zona pellucida removal. Embryos were cultured for 48h in CMRL (11530037, Thermo Fisher Scientific) supplemented with 1 B27 (17504001, Thermo Fisher Scientific), 1 N2 (made in-house), 1 penicillinstreptomycin (15140122, Thermo Fisher Scientific), 1 GlutaMAX (35050-038, Thermo Fisher Scientific), 1 sodium pyruvate (11360039, Thermo Fisher Scientific), 1 essential amino acids (11130-036, Thermo Fisher Scientific), 1 non-essential amino acids (11140-035, Thermo Fisher Scientific) and 1.8mM glucose (G8644, Sigma) supplemented with 20% foetal bovine serum5,28. Embryos were incubated with 25ngml1 Activin-A, 200nM LDN, 50ngml1 BMP6, 20M DAPT or DMSO for 48h. For E4.5, E5.5 and E5.75 collections, embryos were dissected directly from the uteri and fixed for analysis. For E5.0 collection, embryos were dissected from the uteri, and Reicherts membrane was removed before culturing or 36h with relevant small molecules as described above.

Shef6 human ES cells (R-05-031, UK Stem Cell Bank) were routinely cultured on 1.6% v/v Matrigel (354230, Corning) in mTeSR1 medium (85850, STEMCELL Technologies) at 37C and 5% CO2. Cells were passaged every 35days with TrypLE Express Enzyme (12604-021, Thermo Fisher Scientific). The ROCK inhibitor Y-27632 (72304, STEMCELL Technologies) was added for 24h after passaging. Cells were routinely tested for mycoplasma contamination by polymerase chain reaction. To convert primed human ES cells to RSeT or PXGL naive conditions, cells were passaged onto mitomycin-C inactivated CF-1 MEFs (3103 cellscm2; GSC-6101G, Amsbio) in human ES cell medium containing Dulbeccos modified Eagle medium (DMEM)/F12 supplemented with 20% Knockout Serum Replacement (10828010, Thermo Fisher Scientific), 100M -mercaptoethanol (31350-010, Thermo Fisher Scientific), 1 GlutaMAX (35050061, Thermo Fisher Scientific), 1 non-essential amino acids, 1 penicillinstreptomycin and 10ngml1 FGF2 (University of Cambridge, Department of Biochemistry) and 10M ROCK inhibitor Y-27632 (72304, STEMCELL Technologies). For RSeT conversion, cells were switched to RSeT medium (05978, STEMCELL Technologies). Cells were maintained in RSeT and passaged as above every 46days. For PXGL conversion, previously described protocols were used97. Briefly, cells were cultured in hypoxia and medium was switched to chemically Resetting Media 1 (cRM-1), which consists of N2B27 supplemented with 1M PD0325901 (University of Cambridge, Stem Cell Institute), 10ngml1 human recombinant LIF (300-05, PeproTech) and 1mM valproic acid. N2B27 contains 1:1 DMEM/F12 and Neurobasal A (10888-0222, Thermo Fisher Scientific) supplemented with 0.5 B27 (10889-038, Thermo Fisher Scientific) and 0.5 N2 (made in-house), 100M -mercaptoethanol, 1 GlutaMAX and 1 penicillinstreptomycin. cRM-1 was changed every 48h for 4days. Subsequently, medium was changed to PXGLN2B27 supplemented with 1M PD0325901, 10ngml1 human recombinant LIF, 2M G6983 (2285, Tocris) and 2M XAV939 (X3004, Merck). PXGL cells were passaged every 46days using TrypLE (12604013, Thermo Fisher Scientific) for 3min, and 10M ROCK inhibitor Y-27632 and 1lcm2 Geltrex (A1413201, Thermo Fisher Scientific) were added at passage for 24h.

For small-molecule experiments, primed or PXGL human ES cells were plated into ibiTreat dishes at normal passage densities. Forty-eight hours after passage, medium was changed to N2B27 supplemented with 25ngml1 Activin-A, 2M A83-01, 50ngml1 BMP6, 200nM LDN or 20M DAPT. Plates were then fixed for 20min in 4% paraformaldehyde for downstream analysis. For 3D culture of primed human ES cells, 30,000 cells were resuspended in 200l of ice-cold Geltrex and the resulting mix was plated into a single well of an 8 -well ibiTreat dish. Geltrex was polymerized by placement at 37C for 10min. Two-hundred microlitres of mTeSR1 with ROCK inhibitor Y-27632 was added after polymerization. Twenty-four hours later, the medium was changed to N2B27 (10M DAPT). Medium was refreshed 24h later, and the plate was fixed in 4% paraformaldehyde for 30min after a total of 48h in experimental conditions. Conditioned medium experiments were performed as described previously48. Briefly, 80l of ice-cold Geltrex was added to an 8 -well ibiTreat dish to create a 100% Geltrex bed. This was polymerized at 37C for 4min. A total of 1103 cellscm2 primed human ES cells were then added onto this bed in DMEM/F12 and allowed to settle for 15min. After this, medium was carefully switched to conditioned medium (described below) with 5% Geltrex (v/v) and 10M ROCK inhibitor Y-27632. Conditioned medium with 5% Geltrex was refreshed daily for the next 2days, and the resulting spheroids were fixed after a total of 72h.

YSLC differentiation was carried out as published48. Briefly, Shef6 human ES cells cultured in RSeT medium for at least 2weeks were plated onto ibiTreat dishes at 1103 cellscm2 in RSeT medium with 10M Y-27632. Medium was changed the next day to ACL differentiation medium consisting of N2B27 supplemented with 5% v/v Knockout Serum Replacement, 100ngml1 Activin-A, 3M CHIR99021 (University of Cambridge Stem Cell Institute) and 10ngml1 human recombinant LIF. Medium was refreshed every 48h, and 2M A83-01, 200nM LDN or 20M DAPT was added to ACL medium for 48h from either day 2 to day 4, followed by fixation, or day 4 to day 6 followed by fixation. For conditioned medium experiments, at day 6 cells were washed three times with phosphate-buffered saline and then mTeSR Plus medium (100-0276; STEMCELL Technologies) was added for 24h. Medium was collected from YSLCs and passed through a 0.45-m filter (16555, Sartorious), and stored for up to 1week at 4C.

CD1 mouse ES cells (generous gift from Prof. Jennifer Nichols (Stem Cell Institute, University of Cambridge, UK)) were routinely cultured on gelatin-coated (G7765, Sigma Aldrich) dishes in N2B27 supplemented with 1m PD0325901, 3m CHIR99021 and 10ngml1 mouse Lif (University of Cambridge, Stem Cell Institute). Medium was changed every 48h, and cells were passaged every 35days using trypsinethylenediaminetetraacetic acid (25300062; Life Technologies). For experiments, cells were passaged as normal into ibiTreat dishes. The following day, medium was switched to either N2B27+2iLif, N2B27, or N2B27+200nM LDN. Medium was refreshed after 24h, and cells were fixed after 48h.

Embryos were fixed in 4% paraformaldehyde, permeabilized in 0.1M glycine with 0.3% Triton X-100 and placed in blocking buffer containing 1% bovine serum albumin and 10% foetal bovine serum. Primary antibodies were diluted in blocking buffer and added overnight at 4C. Fluorescently tagged secondary antibodies were added for 2h at room temperature. Primary antibodies used in this study are as follows: mouse monoclonal anti OCT3/4 (sc5279, Santa Cruz; 1:200 dilution), rat monoclonal anti SOX2 (14-19811-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti NANOG (AF1997 R&D Systems; 1:500 dilution), rabbit monoclonal anti GATA6 (5851, Cell Signaling Technology; 1:2,000 dilution), goat polyclonal anti GATA6 (AF1700, R&D Systems; 1:200 dilution), mouse anti monoclonal Cdx2 (MU392-UC, Biogenex; 1:200 dilution), goat polyclonal anti CER1 (AF1075, R&D Systems; 1:250 dilution), rat monoclonal anti Cerebus1 (MAB1986, R&D Systems; 1:200 dilution), rabbit monoclonal anti Phospho-Smad1(Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (13820T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti Smad2.3 (8685T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti-cleaved caspase 3 (9664, Cell Signaling Technology; 1:200 dilution), mouse monoclonal anti Podocalyxin (MAB1658, R&D Systems; 1:500 dilution), goat polyclonal anti Brachyury (AF2085, R&D Systems; 1:500 dilution), rat monoclonal anti GATA4 (14-9980-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti AP2-gamma (AF5059, R&D Systems; 1:500 dilution), goat polyclonal anti Otx2 (AF1979, R&D Systems; 1:1,000 dilution) and Alexa Flour 594 Phalloidin (A12381, Thermo Fisher Scientific; 1:500 dilution).

Immunofluorescence images were captured on a Leica SP8 confocal and processed and analysed using Fiji (http://fiji.sc). Epiblast, hypoblast and CER1-positive cell numbers were manually counted using the multi-point cell counter plugin. Quantification of trophectoderm was performed using Imaris software (version 9.1.2) using the spots tool with manual curation. To quantify n/c SMAD2.3 in human and mouse embryos, the central three planes of individual cells were used to generate a three-plane z-stack. Individual 4,6-diamidino-2-phenylindole (DAPI)-positive nuclei were used to generate a nuclear mask using the Analyze Particles function on either the DAPI or lineage-associated transcription factor channel. The adjacent cytoplasmic area was drawn individually for each nucleus and the mean fluorescence of each region was measured, and the ratio computed. When embryos were stained with E-Cadherin, the membrane was delineated to allow for cytoplasmic region of interest determination. When embryos were stained with podocalyxin, the cytoplasmic region of interest was drawn to ensure delineation of a region captures suitable intra-cellular variation allowing for valid normalization. Measurements were computed on raw SMAD2.3 signal. To quantify pSMAD1.5.9 nuclear intensity, a nuclear mask generated on a central three-plane z-stack for each nucleus, and mean fluorescence values were measured. Within each three-plane z-stack, a background fluorescence taken adjacent to or within a cavity of the embryo was used for background normalization ((frac{text{mean nuclear intensity}}{1+text{mean background intensity}})). Background was normalized to (that is, to provide a comparable signal-to-noise ratio) rather than subtracted to account for the variability in laser penetration between experiments and z-planes. In stem cell experiments, nuclear masks were generated, mean fluorescence was measured, and all values were normalized to a control (DMSO) value of 1. To calculate the percentage of cleaved caspase-3-positive or CER1-positive cells, individual cells were manually counted using the cell counter plugin and presented as a percentage of all DAPI-negative or GATA6-positive cells. For 3D spheroid classification, the total number of structures was counted manually using the Cell Counter plugin, and each was assigned to a class of spheroid. For conditioned medium 3D spheroid quantifications, the central three planes of individual spheroids were used to generate a nuclear mask on the DAPI channel, and the mean nuclear pSMAD1.5.9 signal was quantified along with the signal of an acellular region for background normalization. To generate figures, images were processed by generating z-stacks of approximately five to ten planes to allow for visualization of embryo topology with cells on disparate planes followed by consistent adjustment of brightness and contrast.

No statistical method was used to pre-determine sample sizes. Sample sizes are similar to previous publications7,10,24. For characterization of normal development, embryos lacking any of the three lineages were excluded. Multiple SMAD fluorescence intensities were taken per lineage per embryo. All embryos were included in functional experiments and each cell type count is taken from individual embryos. All stem cell experiments were performed independently at least twice. Investigators were not blinded to group allocation during the experiment or analysis, as blinding would not have been possible due to medium preparation and changing requirements. Group allocation was not performed randomly; rather, based on visual assessment of embryos, investigators attempted to ensure balanced distributions of blastocysts/implanting embryos assessed as expanded with nice inner cell masses versus embryos that appeared delayed or with visible cell death across experimental groups. Statistical tests, except the Bayesian distribution model, were performed in Prism 9 (GraphPad), and where relevant, two-sided tests were used. Normality was tested with a ShapiroWilk test. Bayesian distribution modelling, which is suited to the small sample sizes used in human embryo studies, was used as a supplemental tool to assess how each small-molecule treatment affected the distribution of cell number. To do this, the brms R package was used98,99, with the assumption of a Poisson distribution and the Control counts set to inform the priors and be used as reference. Brms default Markov chain Monte Carlo settings were used. Coefficient credible intervals were either below 0.33, denoting a decrease in the distribution compared to control, or above 0.33, indicating an increase in the distribution compared to control. Credible intervals that bridged this range indicate no significant difference. All coefficients and credible intervals in addition to MannWhitney test P values are presented in Supplementary Table 8. For data presentation, box plots encompass the 25th to 75th percentile in the box, with the median marked by the central line, and the mean marked by a cross. The minimum and maximum are marked by the whiskers. For violin plots, the dashed line marks the median value and dotted lines mark the 25th and 75th percentiles. For summary plots (for example, Fig. 2h,i), the mean standard error of the mean is plotted.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Distinct pathways drive anterior hypoblast specification in the implanting human embryo - Nature.com

Confronting IVF: Human Embryos Are Persons With a Right to Life – Walter Bradley Center for Natural and Artificial Intelligence

On Sunday I posted on the Alabama Supreme Courts ruling that frozen embryos are considered children (i.e., minor persons) under Alabama law. The decision is well-reasoned and legally correct, and it highlights the profound ethical issues that accompany in vitro fertilization (IVF) of human beings. In that post, I also commented on Yale neurologist Steven Novellas uninformed opinion on the legal decision.

In that same post, Frozen embryos are not people, Novella offers an opinion on the ethics of IVF that is more thoughtful. I disagree with his conclusion that human embryos are not persons from an ethical standpoint. Ill examine his view step by step, and then provide my own.

Novella:

I [lay] out the core question here when does a clump of cells become a person? Standard rhetoric in the anti-abortion community is to frame the question differently, claiming that from the point of fertilization we have human life. But from a legal, moral, and ethical perspective, that is not the relevant question. My colon is human life, but its not a person. Similarly, a frozen clump of cells is not a child.

What is a potential human?

Unlike many people who deny the personhood of very young human beings, Novella recognizes the obvious scientific fact that human life begins at fertilization and that zygotes/embryos/fetuses/newborns are human beings. They are not potential humans.

A sperm and an egg separately constitute a potential human. But when they unite, the result is a human being from the moment of fertilization. Human beings are not defined by the number of cells in the body be it one cell or 30 trillion cells. A big complex human being is not more human than a small simple human being.

There is no actual debate about this the basic biology of human reproduction was understood in the early 19th century, and any doctor or scientist who denies the humanity of a human being in the womb is either ignorant or deliberately misrepresenting science to advance an ideological agenda. I have explained the scientific fact that human life begins at fertilization here.

And the colon? Novellas invocation of his colon in this debate is nonsense. A colon is an organ its a part of a person, not a whole person. A colon is not a human life. An embryo is a human life, even though he or she is much simpler than Dr. Novellas colon or any other part of an adults body.

This is just basic biology, which Dr. Novella should know.

What is a person?

There are two issues in the IVF debate the question of humanity of the embryo and the question of the personhood of the embryo. They are different questions. The fact that an embryo is a human being is not debatable. But the question Is an embryo a person is another matter, and it is debatable.

What is a person? Novella believes that an embryo is a potential person:

those cells have the potential to become a person. But the potential to become a thing is not the same as being a thing. If allowed to develop those cells have the potential to become a person but they are not a person.

So, according to Novella, what confers personhood on a human being? He states:

The real debate comes down to ethical philosophy and legal theory. How do we balance these various facts:

A human embryo is a human, but not sentient.

Sentience and personhood develops gradually throughout the pregnancy.

Fetuses are dependent on the life of their mother until they develop sufficiently to be viable outside the womb.

Pregnancy is a serious biological process with significant implications for the life of the mother.

Sentience and independence

Novella equates personhood with sentience and independence. Sentience and independence are certainly characteristic of most persons we know (including ourselves), but they are not satisfactory criteria for personhood.

A newborn baby, a person with severe mental handicaps, and a person in a deep coma all lack appreciable sentience and independence but they are undoubtedly persons. Using sentience and independence as fundamental criteria for personhood even implies that temporary unconsciousness such as non-sentience during deep sleep or dependence on others such as being critically ill renders us transient non-persons. In fact, we are persons even when we are non-sentient and dependent on others.

Even more disturbing is the fact that gradations of sentience have long been used to deny basic human rights to categories of people based on real or perceived cognitive differences. An illiterate man is, in a very real sense, less sentient than a literate genius, but is he less of a person?

So what, exactly, is a person? Many different definitions have been offered, but it seems to me that there is one continuous thread that runs through what it means to be a person: a person is a human being who has rights and who is entitled to respect. Certainly, to deny a human being any respect and to deny him all rights is to deny his personhood in a fundamental way.

Most rights of persons are linked to particular acquired skills or milestones in the persons life the right to drive a car, the right to vote, the right to keep and bear arms, etc. Yet there is one right that is not linked to skills or milestones, and it is in fact the right on which all other rights depend the right to life. If I am a person with the right to drive and vote and so on, but not the right to life, my life can therefore be taken from me arbitrarily at the whim of another. Then I really dont have any rights at all, and I am not treated as a person. All other rights are meaningless without the right to life.

The right to life is the indispensable cornerstone of all rights, and the right to life is the cornerstone of personhood. Thus a person is a human being with a right to life.

Are all human beings persons? Or are there two classes of humans?

This is the crux of the debate about IVF, abortion, and other life issues: are all human beings persons with an unalienable right to life? Those of us in the pro-life movement answer that question emphatically in the affirmative. Those who support the destruction of human beings in the womb or in the petri dish, etc., believe there are two classes of human beings: those with the right to life, and those without the right to life. This is the issue at hand.

It is certainly the case that in pregnancy and in the production of frozen embryos there are other rights involved the rights of the mother, the rights of the parents (although, of course, to refer to mother and parents implicitly affirms that the unborn are children, not lumps of cells). In sorting out these various rights the right to bodily autonomy, the right to dispose of property, etc. the right to life always takes precedence.

The right to life is the cornerstone of all rights, and even when the rights of the mother or parents are significant and generally worthy of protection, they do not trump the right to life of the young human being the young person in the womb or in the petri dish or in the nursery.

The implications of IVF

A deeply troubling social and moral issue arises with IVF as well. IVF is the industrial manufacture of human beings. The brave new world that is dawning on us has risks and horrors that should chill us. IVF provides the opportunity to screen embryonic human beings for genetic traits (which is already being done), and this technology can and will be used in the future to breed human beings with certain desired traits obsequious servants, aggressive warriors, attractive sex slaves, and compliant organ donors.

We are learning to mass produce human beings, and although the technology thus far has been used mainly for the sympathetic goal of providing childless couples with children, only a naif would believe that it will end here. The industrial manufacture of human beings opens the door to evil on a scale that is the stuff of nightmares.

IVF is an ethically problematic technology with horrifying implications. If we are to survive this brave new world of mass-produced children, conceived not via conjugal love of a husband and wife in a family but via a pipette in a human assembly line, we must affirm that human life begins at fertilization, and that all human beings are persons with the right to life and deserving of respect. This is the essence of the pro-life movement and the philosophy of human exceptionalism. It is stated most cogently in the Christian view that we are created in Gods image. It is that Image that confers personhood and the unalienable right to life on each of us, from fertilization to natural death.

You may also wish to read: Are IVF human embryos children? A recent court decision Neurologist Steven Novella claims that the Alabama Supreme Court ruling that they are children under the law essentially referenced god The ruling not only did not reference God, it was meticulously based on precedent. So those who seek to remove protection from IVF embryos must lobby for that.

See more here:
Confronting IVF: Human Embryos Are Persons With a Right to Life - Walter Bradley Center for Natural and Artificial Intelligence

In light of the Alabama court ruling, a look at the science of IVF : Short Wave – NPR

Blastocyst illustration. A blastocyst is a hollow ball of cells with a fluid centre formed after several divisions of a fertilised cell (zygote). The inner cell mass (purple) contains the cells that will form the embryo proper, the embryonic stem cells (ESCs). Kateryna Kon/Science Photo Library/Getty Images hide caption

Blastocyst illustration. A blastocyst is a hollow ball of cells with a fluid centre formed after several divisions of a fertilised cell (zygote). The inner cell mass (purple) contains the cells that will form the embryo proper, the embryonic stem cells (ESCs).

Since the first successful in vitro fertilization pregnancy and live birth in 1978, nearly half a million babies have been born using IVF in the United States. Since the first successful in vitro fertilization pregnancy and live birth in 1978, nearly half a million babies have been born using IVF in the United States. Reproductive endocrinologist Amanda Adeleye explains the science behind IVF, the barriers to accessing it and her concerns about fertility treatment in the post-Roe landscape.

For more on IVF success rates, check out the Society for Assisted Reproductive Technology's database.

Questions or ideas for a future episode of Short Wave? Email us at shortwave@npr.org we'd love to hear from you!

Listen to Short Wave on Spotify, Apple Podcasts and Google Podcasts.

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This episode was produced by Berly McCoy and Rebecca Ramirez. It was edited by Brit Hanson and Rebecca Ramirez. Brit checked the facts. The audio engineer was Josh Newell.

See the original post:
In light of the Alabama court ruling, a look at the science of IVF : Short Wave - NPR

Runx1+ vascular smooth muscle cells are essential for hematopoietic stem and progenitor cell development in vivo – Nature.com

A subpopulation of subaortic mesenchyme in the AGM co-expresses NG2 and Runx1

We examined the expression of PC/vSMC markers in the dorsal aorta of E10.5 and E11 mouse embryos. Wholemount immunostaining and immunohistochemistry on frozen sections were performed using PC/vSMC markers NG2 or SMA with CD31, an endothelial and HSPC marker (TableS1). Imaging analysis showed that NG2+SMA+CD31- vSMCs surround NG2-SMA-CD31+ endothelial cells (Figs.1a, S1a, b), confirming previous reports27. Further to its expression in hematopoietic and hemogenic endothelial cells, Runx1 was also detected in the sub-aortic mesenchyme22,23. Therefore, we hypothesised that at least some of these cells also express NG2. We first confirmed that both intra-aortic hematopoietic cell clusters (IAHCs) (Fig.1b, stars) and hemogenic endothelial cells (Fig.1b, arrowheads) are Runx1+; we also identified a subpopulation of NG2+ PC/vSMCs, mainly located in the ventral aspect of the dorsal aorta, that also express Runx1 (Fig.1b, arrows). Other Runx1+ cells in the perivascular area do not express NG2 (Fig.1b). Finally, we confirmed our recent study28 that some cells around the notochord express NG2 in the trunk (Fig.S1a, circle). However, these peri-notochord cells do not express SMA, CD31 (Fig.S1a, circle) nor Runx1 (Fig.S1ef). To confirm the presence of NG2+Runx1+ cells in the E11 AGM, we used Runx1-IRES-GFP mouse embryos29. In these GFP knock-in mice, GFP intensity correlates with Runx1 expression level. Flow cytometric analysis showed the presence of a distinct population of NG2+Runx1(GFP)+ cells in the AGM (Fig.1c). These cells first appear at E10, in line with the presence of Runx1 in mesenchymal cells30 and importantly, their frequency peaks at E10.5 (Fig.1d). Together, these data show that in the AGM, a subset of the sub-aortic mesenchyme expresses both NG2 and Runx1 and that the highest frequency of these cells coincides with the onset of HSC generation at E10.5.

a Three-dimensional (3D) wholemount immunostaining with SMA, CD31 and NG2 of E10.5 (3138 somite pairs (sp)) WT dorsal aorta; b NG2 and Runx1 expression on single plane wholemount WT E10.5 sections. NG2+Runx1+vSMCs (arrows), hemogenic endothelial cells (arrowheads) and intra-aortic hematopoietic clusters (IAHCs, stars) (TableS1); c Representative example of flow cytometric analysis of NG2+Runx1(GFP)+ (green box) in E10.5 Runx1-IRES-GFP AGM and E10.5 WT control. d Percentages of NG2+Runx1(GFP)+ cells in E9 (21-25sp) body (n=6), E10/E10.5/E11 AGMs (n=8/7/7), N=5, Kruskal-Wallis and Dunns post-hoc test. e Representative examples of wholemount 3D-images showing SMA, CD31 and NG2 in E10.5 cKO dorsal aortae; f SMA, Runx1 and CD31 immunofluorescence of E11 WT and cKO transversal frozen sections; n=WT/cKO: 2/2, N=2. g cKit and CD31 wholemount 3D-images in E10.5 WT and cKO AGM; h Number of intra-aortic hematopoietic clusters (IAHCs) in E10.5 AGM; n=WT/KO: 5/4, N=4. Number of colony forming unit-culture (CFU-C) in i E10.5 (31-38sp) AGM; n=WT/HET/KO: 14/10/5 embryos; N=7 and j E11 (4352sp) AGM; n=WT/HET/KO: 22/8/19 embryos; N=11; one-way ANOVA and Tukeys post-hoc test (TableS2). k Percentages of donor cell chimerism 4-months post-transplantation of 6 E11 WT (NG2+/+;Runx1fl/+or NG2+/+;Runx1fl/fl), 7 HET (NG2-Cre;Runx1fl/+) and 6 cKO AGMs (NG2-Cre;Runx1fl/fl) into sub-lethally adult irradiated recipients (1xAGM cells transplanted/recipient; N=4). Each dot represents one recipient. Mice are reconstituted when 5% donor cells are found in the host peripheral blood (dashed line); one-tailed Z score test for two population proportions (TablesS3 and S4). For wholemount staining in a, b, e, g: WT/cKO (N=6/4): SMA (n=9/7), CD31 (n=10/7), cKit (n=3/2), NG2 (n=3/1) and WT Runx1 (n=4) in 3 distinct combinations (TableS1). D = dorsal, V = ventral. N = number of independent experiments; n = number of biological samples (embryos). All data are presented as mean valuesSEM. Source data for d, h, i, j and k are provided as a Source Data file.

Runx1 deletion in endothelial cells impairs HSC emergence in the AGM24,25,26. However, the effect of Runx1 deletion in PC/vSMCs on hematopoiesis in vivo is still unknown. To address this, we examined conditional knock-out (cKO) NG2-Cre;Runx1fl/fl mouse embryos. In previous studies, the NG2-Cre mouse strain revealed a role for pericytes in supporting both fetal liver and adult bone marrow HSC maintenance31,32. Our data shows that E10.5 and E11 cKO embryos do not exhibit visible vascular abnormalities. This was confirmed by the normal expression of CD31, SMA and NG2 (Figs.1e, f, S1c, d) in the AGM. In contrast, SMA+Runx1+ PC/vSMCs with low expression of Runx1 were reduced in the cKO dorsal aorta compared to WT littermate controls (Figs.1f, S1eg). CD31+Runx1+ endothelial cell number and frequency was also decreased (Fig.S1g). Furthermore, CD31+cKit+ IAHC numbers were significantly reduced by three-fold (p=0.02) (Fig.1g, h). Hematopoietic progenitor (HP) assays were performed to test if hematopoietic function was affected. All HP numbers were significantly reduced in cKO AGMs at both E10.5 (Fig.1i, TableS2) and E11 (Fig.1j, TableS2). To test whether definitive HSCs were also affected, we performed HSC assays in vivo. At 1- and 4-months post-transplantation of AGM cells into sub-lethally irradiated mice, chimerism and multilineage reconstitution were examined by flow cytometry in the peripheral blood. Compared to the WT littermate control group, in which 66.7% (4 out of 6) recipients were reconstituted, only 14.3% (1 in 7, p=0.025) and 16.7% (1 in 6, p=0.040) mice injected with heterozygous or homozygous cKO AGMs, respectively, were reconstituted over the long term (Fig.1k, TablesS34). These findings indicate that the absence of Runx1 in aortic NG2+ cells impairs HSC generation and/or maintenance and HP development in the AGM.

To test whether NG2+ cells contribute directly to hematopoietic lineages, we isolated NG2+ and NG2+Runx1(GFP)+cells from E11 WT and Runx1-IRES-GFP AGMs, respectively, and seeded them in methylcellulose. In parallel, NG2- or NG2-cKit+ cells were sorted as controls. HPs were exclusively found in the NG2- cell fractions. Neither NG2+ cells (Fig.S2a) nor NG2+Runx1(GFP)+cells (Fig.S2b) gave rise to hematopoietic cell colonies in vitro (TableS5). To further assess whether NG2+ cells are hematopoietic precursors, we crossed NG2-Cre mice with a knock-in reporter mouse line in which tdTomato is preceded by a transcriptional stop flanked by two loxP sites under the Rosa26 promoter. In these mice, NG2+ cells and their progeny are tdTomato+. E11 AGM-derived tdTomato+ and tdTomato- cells were sorted and seeded in methylcellulose. HPs were only found in the tdTomato- cell fraction (Fig.S2c, TableS5) reinforcing the observation that NG2+ cells and their progeny do not contribute to hematopoietic lineages at this stage. Flow cytometric analysis confirmed the presence of tdTomato in a subset of NG2+ cells in the E11 AGM (Fig.S2d), validating our mouse model, while no overlap was found between tdTomato and CD45, a hematopoietic cell marker (Fig.S2e). We next performed immunohistochemistry on NG2-Cre;tdTomatofl/+ frozen sections and confirmed the expression of tdTomato in a subset of SMA+ cells (Fig.S2f) in the E11 AGM. CD31+ cells did not express tdTomato (Fig.S2g). Further analysis revealed that cells expressing hematopoietic markers F4/80 and CD45 do not co-express NG2 nor SMA (Fig.S2h). Together, these data indicate that NG2+ cells do not contribute to the AGM HSPC pool and suggest that NG2+Runx1+ PC/vSMCs act as a supportive niche to maintain hematopoietic activity in the AGM.

In the early developing embryo, HSPCs reside in other intra-embryonic and extra-embryonic hematopoietic organs such as the head, fetal liver (FL), placenta and yolk-sac (YS). Flow cytometric analysis of these organs harvested from Runx1-IRES-GFP mouse embryos also confirmed the presence of NG2+Runx1(GFP)+ cells (Fig.S3a, b). We next performed in vitro HP functional assays with cells harvested from all organs and genotypes of NG2-Cre;Runx1fl at both E10 and E11 developmental stages. No significant differences were found when comparing the total CFU-C numbers between genotypes in most organs (Fig.S3c, d, TablesS67). A significant increase of total number of CFU-C was observed in E10 AGM in both heterozygous and cKO mouse embryos (Fig.S3c). When analyzed individually, a significant increase in the number of erythroid colonies was detected in the cKO compared to WT littermate (p=0.0149) (TableS6). Likewise, a 2.8-fold increase in the number of erythroid colonies was detected in the E11 cKO head compared to the WT littermate (p=0.01), while the total number of CFU-C in the E11 head remained unchanged (TableS7). Moreover, we found a significant decrease in both CFU-GM (p=0.016) and CFU-GEMM (p=0.039), between WT and cKO YS (TableS7), possibly due to the defect found in the E11 AGM.

To test whether HSC activity increases in the FL due to the possible migration of AGM HSCs, E11 FL cells from all genotypes were transplanted into sub-lethally irradiated recipient mice. Neither the donor chimerism nor the percentage of reconstituted mice by donor cells showed changes between the groups (Fig.S3e). Compared to NG2+/+;Runx1fl/+ WT littermates, in which 70% of recipients (7 out of 10) were reconstituted, mice injected with NG2-Cre:Runx1fl/+ heterozygous or NG2-Cre:Runx1fl/fl cKO E11 FL showed similar reconstitution over the long term, with 67% (2 out of 3, p=0.348) and 60% (3 out of 5, p=0.421) reconstituted mice, respectively (Fig.S3e, TablesS34). Since the deletion of Runx1 in NG2+ cells only affects HSPCs in the AGM, immunohistochemistry on WT embryonic head and placenta was performed to localise NG2+Runx1+ cells. The rare NG2+Runx1+ double positive cells identified did not seem to be perivascular (Fig.S3f, stars). In line with this observation, we found that Runx1 and SMA do not overlap when NG2 and SMA were expressed in PC/vSMCs (Fig.S3f, arrowheads). Instead, the head contains few NG2+SMA- that are F4/80+, suggesting that NG2+Runx1+ cells are macrophages (Fig.S3f, arrowhead). Overall, our data shows that the deletion of Runx1 in NG2+ cells only affects selective HSPC subsets in non-AGM hematopoietic organs in the E11 mouse embryo.

To better understand the role of Runx1 in the HSC-generating microenvironment, single-cell RNA-sequencing (scRNA-seq) on NG2+/+;Runx1fl/+ E11 AGM was performed. We used graph-based clustering and known marker distribution to define and investigate the gene expression profiles of various populations that reside in the E11 AGM and identified eight populations of interest (Fig.2a, b). The co-expression of Cspg4 (NG2) and Acta2 (SMA) in the PC/vSMC population was confirmed (Fig.2c). This population is also enriched in Rgs5, Pdgfrb and Pdgfra in line with our previous work28, and a subset of these cells express Runx1 (Fig.2c, d), confirming our imaging and flow cytometric analysis. The expression of Mcam (CD146 or S-ENDO1), a pericyte/vSMC precursor marker recently identified in a subset of NG2+ cells in the E11 AGM21 and upregulated in AGM hematopoiesis supportive stromal cell lines19, was detected in a subset of PC/vSMCs, partially overlapping with Runx1+ cells (Fig.2c, d). However, Mcam was mainly enriched in endothelial cells (ECs) and also in subpopulations of hemogenic endothelial cells, including those entering endothelial-to-hematopoietic transition (HEC/EHT), IAHCs and SNS cells (Fig.2d), confirming published work including ours28,33. Immunostainings with CD146 and CD31 on E11 WT AGM frozen sections further validated our sequencing analysis at the protein level: both CD31+ endothelial cells (Fig.2e, f, arrows) and SMA+ PC/vSMCs (Fig.2f, stars) are CD146+. Importantly, Pecam-1 (CD31) expression in PC/vSMCs was low/negative in our scRNA-seq data (Fig.2c, d), in line with our immunohistochemistry, confocal imaging, and our recent published work28. Other genes expressed by hematopoietic and hemogenic/endothelial cells such as Adgre1 (F4/80), Mrc1 (CD206), Cdh5 (VE CADHERIN), Tek (TIE-2), CD34, CD93, Pdgfb, Sox7, Sox17, Sox18, Gfi1b, and Itga2b (CD41) were not expressed in PC/vSMCs (Fig.2d). These genes were used to distinguish populations of macrophages (MPs), IAHCs, HEC/EHT, and ECs (Fig.2ad). Erythroid cellsand erythroid progenitors (Ery/EryP; Gypa/CD235+), SNS (Gata3+) and skeletal muscle progenitors (SkMP; MyoD1+, Cdh15+) were also identified (Fig.2ad). Kit was expressed in all IAHCs and in a subset of PC/vSMCs, while Kit expression in HEC/EHTs was low (Fig.2d). Altogether, these data show that we successfully captured multiple cell types that comprise the E11 AGM, including a population of Runx1+ PC/vSMCs which constitutes 19.7% of all NG2+Acta2+ PC/vSMCs cells. Furthermore, the transcriptome of Cspg4+Runx1+ non-hematopoietic non-endothelial PC/vSMCs was found to partially overlap with that of the Cspg4+Mcam+ PC/vSMC precursors previously described21.

a t-SNE plot highlighting eight populations of interest identified in the E11 WT AGM. Each dot represents one cell and colours represent cell clusters as indicated. The number of cells in each population is shown in brackets. MP (macrophages); Ery/EryP (erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enter endothelial-to-hematopoietic transition); EC (endothelial cells); SNS (sympathetic nervous system); SkMP (skeletal muscle progenitors), PC/vSMC (pericytes/vascular smooth muscle cells, NG2+Acta2+). Other cells (OC) are coloured in grey. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Zoom into PC/vSMC cluster (black rectangle) further show the presence or the absence of selected genes that characterise this population and confirms the presence of Runx1 in a subset of cells. d Violin plots showing distribution of expression for selected genes that contributed to the identification of cell clusters. Immunohistochemistry on frozen E11 WT sections stained with eCD146/CD31/DAPI and fCD146/SMA/DAPI, n=2 samples tested, N=2 independent experiments. Arrows: vascular cells, asterisks: perivascular cells. DA: dorsal aorta, CV: cardinal veins, NC: notochord. Source data for e (first column,20X) is provided as a Source Data file.

Our scRNA-seq analysis revealed that not all Cspg4+Runx1+ cells in the E11 AGM express Acta2 (Fig.3a, b). We therefore investigated if Cspg4+Runx1+Acta2 cells are PC/vSMCs which had not yet acquired Acta2 expression. Differential expression analysis of Acta2+ versus Acta2 cells within the NG2+Runx1+ cell population in the WT AGM revealed that markers of sclerotome-derived vSMCs such as Sox9, Pax1, Pax9 and Col2a134 are among the highest upregulated genes in Cspg4+Runx1+Acta2 cells (Fig.3c). In contrast, Cspg4+Runx1+Acta2+ cells are enriched in genes that identify more mature pericytes such as Acta2, CD248, Mcam, Rgs5 or Pdgfrb (Fig.3c), some of which are potential Runx1 target genes (star). Pdgfra and Ptn genes were recently associated with Runx1+ subaortic (non-smooth muscle) mesenchymal cells with possible role in hematopoiesis in the E10.5 AGM of the mouse embryo35. Our scRNA-seq analysis show that, in E11 AGM, Pdgfra and Ptn are also expressed in Cspg4+Runx1+ cells with no significant difference between Acta2+ and Acta2 (Fig.3c). Further analysis showed that Gene Ontology (GO) biological processes significantly enriched in Cspg4+Runx1+Acta2+ cells include smooth muscle cell chemotaxis and migration, collagen-activated signalling pathway, neural crest cell differentiation and regulation of BMP signalling (Fig.3d), previously shown by our laboratory to control in vivo HSPC generation in the mouse AGM28. In Cspg4+Runx1+Acta2 cells, significantly enriched GO biological processes include mesenchymal stem cell differentiation and cartilage and bone development (Fig.3e), consistent with the sclerotome origin of these cells.

a t-SNE plots showing the distribution of Runx1 and Acta2 expression in NG2+Runx1+ cells in the WT E11 AGM after excluding all other (grey) cells found in the Fig.2a. b Zoom into NG2+Runx1+ cluster (black rectangle) shows the presence or the absence of Acta2. c Heatmap showing the expression of Cspg4 and Runx1 and 15 selected genes out of 25 top significantly upregulated genes in WT NG2+Runx1+Acta2+ cells (upper half) and NG2+Runx1+Acta2- cells (bottom half) at single cell level; *Runx1 potential target genes. Pdgfra and Ptn genes were next added to inform their expression in both populations. Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly upregulated in both dWT NG2+Runx1+Acta2+ and eNG2+Runx1+Acta2- cells. f t-SNE of WT E11 AGM cells, overlaid with principal pseudotime curve inferred by Slingshot, predicting a lineage from NG2+Runx1+Acta2- cells to NG2+Runx1+Acta2+ cells. g WT NG2+Runx1+cells arranged in pseudotime (x-axis) based on the inferred curve. Y-axis represents log normalised gene expression.

Indeed, PC/vSMCs in the AGM have been shown to originate from the sclerotome and display markers of this compartment at least during the early phases of mural cell recruitment36. A recent study showed that the maturation of sclerotome-derived vSMCs in the mouse AGM depends on a transcriptional switch from a sclerotome signature with the repression of Pax1, Scx and Sox9, and activation of Acta2 and other vSMC genes34.

To test whether NG2+Runx1+Acta2 cells follow a maturation trajectory towards Cspg4+Runx1+Acta2+ vSMCs, we performed cell lineage inference with Slingshot, a trajectory inference method for scRNA-seq data that can incorporate knowledge of developmental markers. Having defined a cluster of Cspg4+Runx1+Acta2 cells as an origin, Slingshot infers a cell lineage and constructs a pseudotime curve representing that lineage (Fig.3f, arrow). Gene expression along pseudotime shows that sclerotome markers such as Sox9, Pax1 and Pax9 are gradually downregulated while markers of mural cells such as Acta2, Rgs5, Pdgfr, Cnn1, Mcam and CD248, are gradually upregulated in an inferred transition from Cspg4+Runx1+Acta2 to Cspg4+Runx1+Acta2+ cells (Fig.3g). Our scRNA-seq analysis shows that Cspg4+Runx1+ AGM cells display a sclerotome-derived vSMC transcriptomic profile.

We next explored the impact of Runx1 deletion in the hematopoietic niche and its possible effect on PC/vSMCs by performing scRNA-seq of NG2-Cre;Runx1fl/fl cKO E11 AGMs (Fig.4a, b). Cell populations were defined in a similar way to the WT AGM by using graph-based clustering and known marker distribution. This comparison revealed changes in the proportions of the different cell types between WT and cKO AGM, including a significant reduction in the proportion of cells associated with clusters 2 (Ery/EryP), 3 (IAHC), 6 (SNS), and 7 (SkMP) (Fig.4c).

a t-SNE plot showing eight populations of interest found in the E11 cKO AGM. Each dot represents one cell and colours represent cell clusters as indicated. MP(macrophages); Ery/EryP(erythroid/progenitors); IAHC (intra-aortic hematopoietic clusters); HEC/EHT (hemogenic endothelial cells including those that enterendothelial-to-hematopoietic transition),EC (endothelial cells); SNS (sympathetic nervous system); SkMP(skeletal muscle progenitors); PC/vSMC (pericytes/vascular smooth muscle cells, NG2+Acta2+). Other cells (OC)are coloured in grey. The number of cells in each cluster is shown in brackets. b t-SNE plot highlighting the eight populations identified after excluding all other (grey) cells. c Percentage of single live cells found in each E11 AGM sample (cell number/total cells) defined by scRNA-seq in WT (full bars) and cKO (empty bars) AGMs. Colours and numbers correspond to each population defined in a; chi-squared two-tailed test was used for comparison. d Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly downregulated in cKO PC/vSMCs compared to their WT counterparts. Heatmap of ligand-receptor interactions inferred by NicheNet from e WT and f cKO E11 AGM cells. Colour represents the interaction potential score between the 10 top-ranked ligands expressed in ECs and their inferred targets expressed in PC/vSMCs. Ligands and receptors are ordered by hierarchical clustering. g Scatter plots of AUC vs log10(FDR) showing downregulated genes associated with selected GO terms in cKO PC/vSMCs. Red dots represent significantly downregulated genes (FDR<0.05); dashed line shows FDR = 0.05. Gene labels with red borders represent potential Runx1 target genes.

Changes in gene expression between WT and cKO Cspg4+Runx1+ cells were first investigated. We found that genes significantly downregulated in cKO Cspg4+Runx1+ cells were mainly associated with biological processes including translation, oxidative phosphorylation, cellular response to stress and mitochondria-related function (Fig.S4ac). As deletion of Runx1 may have also affected Runx1 PC/vSMCs, the transcriptome of all cKO Cspg4+Acta2+ PC/vSMCs with their WT counterpart was compared. Genes significantly downregulated in cKO Cspg4+Acta2+ PC/vSMCs had significant enrichment of biological processes including translation, smooth muscle cell differentiation, cytoskeleton, vasculogenesis and cell communication (Fig.4d). One pathway essential to vasculogenesis is PDGF-B/PDGFR; we therefore applied NicheNet on our WT scRNA-seq data to predict ligand-receptor interaction between ECs and PC/vSMCs, focusing on PDGF-B-related genes. The highest scoring predicted interaction was between Pdgfb, a growth factor released by ECs, and Nrp1 (Fig.4e), a receptor known to control the differentiation/recruitment of mesenchymal stem cells and the stimulation of smooth muscle cell migration37,38.

The interaction between Pdgfb and Pdgfrb was also amongst the highest scoring interactions in both WT (Fig.4e) and cKO (Fig.4f). Additional interactions involve Edn, Tgfb or Bmp pathways, previously associated with a role in AGM hematopoiesis39,40. Interestingly, in cKO ECs, Pdgfa, another gene from the PDGF family, was no longer in the top 10 ranking ligands (Fig.4f) possibly due to the downregulation of Pdgfra in cKO PC/vSMCs (Fig.4f). Other genes including Des, Angpt1, Gsk3b, Tcf21, Col1a1, Pcna, Ccnd3 and Mcm7, potential Runx1 downstream target genes41, were also significantly downregulated (Fig.4g, red boxes). The reduction inCol1a1 expression suggests changes in the gene profile of the extracellular matrix (ECM). Indeed, additional ECM related genes were significantly downregulated in the cKO PC/vSMCs, such as Sparcl1, Col3a1 and Col5a1 (Fig.4g). Collectively, these data show that the genetic programme of PC/vSMCs in cKO AGM is modified upon Runx1 deletion and this involves changes in molecules that constitute the ECM of the aortic wall.

Endothelial cells share the same basement membrane with PC/vSMCs42. This, coupled with the transcriptomic changes in the cKO PC/vSMCs described above, suggest that the genetic programme of the adjacent ECs may have also been altered by Runx1 deficiency in NG2+ cells. Although the number of endothelial cells in the NG2-Cre:Runx1fl/fl cKO did not significantly change (Fig.4a) and the formation of the dorsal aorta appeared to be unaffected (Fig.1e), we investigated transcriptomic changes in ECs that could affect their function in vivo. As before, we performed differential expression analysis, followed by overrepresentation analysis on genes significantly downregulated in cKO ECs (Fig.5a). Multiple GO biological processes were significantly overrepresented in these genes, with many related to EC development and angiogenesis; proliferation, migration and differentiation; response to hypoxia and fluid shear stress; as well as smooth muscle cell or mesenchymal cell development and hematopoiesis (Fig.5a). Interestingly, we found that Sox18 was the most downregulated gene in cKO ECs (Fig.5b). Col4a1, the most abundant extracellular matrix associated gene, known to co-localise with Sox18 in ECs in the mouse embryo43, was also found within the top 25 downregulated genes (Fig.5b). Sox18 and Col4a1 were the most downregulated genes associated with the blood vessel development GO term, while other gene expression including Cdh5, Pecam1, Sox17, Pdgfb, MCam and Notch were also affected.

a Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly downregulated in cKO ECs compared to their WT counterparts. b Scatter plots of AUC vs log10(FDR) showing downregulated genes associated with selected GO terms including blood vessel development and mesenchymal cells and vSMCs in cKO ECs. Red dots represent significantly downregulated genes (FDR<0.05); dashed line shows FDR=0.05. Sox18 and Ctnnb1 expression in WT ECs in both scRNA-seq (c, EC zoom and t-SNE plots) and bulk RNA-seq post-sort (d, TPM). e Scatter plots of AUC vs log10(FDR) showing downregulated genes associated with selected GO terms including the basement membrane and extracellular matrix in cKO ECs. Red dots represent significantly downregulated genes (FDR<0.05); dashed line shows FDR=0.05. Selected genes that were altered in cKO ECs ine are shown in WT ECs in both scRNA-seq (f, ECand HEC/EHT zoom and t-SNE plots) and bulk RNA-seq post-sort (g, TPM). TPM: transcript per Million mapped reads values.

Genes associated with cell adhesion, regulation of smooth muscle cell proliferation and differentiation, along with mesenchyme development such as Sox18 and Ctnnb1 were also significantly downregulated in cKO EC (Fig.5b, arrow). We confirmed that both Sox18 and Ctnnb1 are expressed by ECs in our single cell datasets (Fig.5c) and next validated their expression in NG2-PDGFR-ckit-CD45-CD31+ Runx1- purified ECs from E11 Runx1-IRES-GFP AGMs (Figs.5d, S5a).

Some significantly downregulated genes associated with blood vessel development such as Loxl2, Hspg2, Col4a2, Col15a1 and Col18a1 (Fig. 5b)are also known to be associated to the ECM. Further analysis of endothelial extracellular matrix encoding genes previously described44 revealed that most of these genes were also significantly downregulated in cKO ECs (Fig.5e). The expression of these genes in WT ECs at single-cell level (Fig.5f) was confirmed post-sort at population-level (Fig.5g) with most genes being highly expressed in ECs only. One of them was Sparc (Fig.5e blue arrow, Fig.5g), a central ECM secreted Ca2+-binding glycoprotein that interacts with many other ECM proteins including Col1 and Col445,46. Among the SPARC family, Sparcl1 (Sparc-like 1), known to bind to Col147, was also found to be significantly downregulated in cKO ECs (Fig.5b, c). Together, these analyses show that Runx1 deficiency in NG2+cells leads to significant transcriptomic changes in endothelial cells including extracellular matrix related genes. We did not detect transcriptional changes in the NG2-Cre;Runx1fl/fl cKO HEC/EHT cell cluster, although this observation is inconclusive due to the low number of cells captured.

Transcriptomic changes in vascular and perivascular cells may have also affected IAHCs. As hematopoietic cells are highly heterogeneous and progenitors were significantly affected (Fig.1), we first explored WT IAHCs in more detail. Previous studies showed that IAHCs are composed of both Runx1+ and Runx1- cells28,48,49 and we were able to confirm this by flow cytometry in Runx1-IRES-GFP AGMs (Fig.S5a). We also confirmed the expression of Runx1 in HEC/EHT and its absence in ECs by flow cytometry in Runx1-IRES-GFP E11 AGMs (Fig.S5a), in line with published work50. To validate their cell identity, we next purified and sequenced 243 Runx1 (GFP)+ and 27 Runx1 (GFP)- IAHCs (NG2-PDGFR- CD31+ckit+) as well as 5822 EC (Runx1-) and 248 HEC/EHT (Runx1+) cells from NG2-PDGFR- ckit- CD45- CD31+ E11 Runx1-IRES-GFP AGMs (Fig.S5a) and performed bulk RNA sequencing (RNA-seq). The purity of the sort was first confirmed (Fig.S5b). While CD45 antibody was not used to isolate IAHCs (Fig.S5a), our bulk RNA-seq data (Fig.S5b) show that not all IAHC cells express Ptprc (CD45) in line with previous studies48,49,51, and seems to be present only when Runx1 is expressed. Next, the identity of all sorted cell populations based on the expression of genes known to be expressed in these cells15,35,48 was confirmed (Fig.S5c). Interestingly, the transcriptomic profile of Runx1 (GFP)+ and Runx1(GFP)- sorted IAHCs appears to be distinct. While CD34, Gata2, Lmo2, Etv6, and Eglf7 are expressed in both Runx1(GFP)+ and Runx1(GFP)- IAHCs at various levels, Adgrg1, Gfi1, Myb and CD44 are mainly found in Runx1(GFP)+ IAHCs (Fig.S5c). Instead, as they also express Tek, Kdr, Eng, Esam and Gata2 (Fig.S5c, d), Runx1(GFP)- IAHCs are at the transcription level, closer to type-1 pre-HSCs52,53 or to recently described CD31+ckithighGata2medium IAHCs that are Runx1-Ptprc-48 with possible (micro)-niche role54. Our analyses confirm the heterogeneity of Runx1(GFP)+/-CD31+C-KIT+IAHCs in the E11 AGM at both protein and transcriptomic levels, and indicate that most IAHC cells captured in our full/unsorted AGM scRNA-seq are Runx1-.

To explore transcriptomic changes between WT and cKO IAHCs, differential expression analysis followed by overrepresentation analysis on genes significantly downregulated in cKO IAHCs was carried out (Fig.6a). Several GO biological processes were significantly overrepresented in these downregulated genes, including ribosome assembly processes, regulation of translation, RNA transport and localisation, and others such as response to DNA damage, gene expression and cellular processes (Fig.6a). In line with this, we found that the top 25 significantly downregulated genes in cKO IAHCs were mostly ribosomal protein coding genes from both Rps and Rpl families. Other genes in the top 25 are known to be required for transcriptional or translational initiation such as Btf3, Pabpc1 and Bclaf1 (Fig.6b). Interestingly, one of the top significantly downregulated genes in the cKO was Sox18 (Fig.6b, arrow), previously reported to be expressed in both IAHCs and ECs in the mouse AGM55 and confirmed here by our WT scRNA-seq data (Fig.2d). Furthermore, Sox18 has been transiently detected during early hematopoiesis in a model of embryonic stem cell differentiation in vitro, controlling early HP proliferation and maturation56. In line with this, further GO analysis revealed that Sox18 is associated with cellular processes including cell maturation, cell differentiation and regulation of stem cell proliferation (Fig.6b). The latter two GO terms are also associated with other genes significantly downregulated in cKO IAHCs such as Hmgb2, encoding a chromatin-associated non-histone protein involved in transcription and chromatin remodelling (Fig.6b). This transcriptomic analysis shows that the deletion of Runx1 in NG2+ PC/vSMCs within the AGM niche significantly alters the genetic programme of IAHCs.

a Barplot of fold enrichment for selected GO biological processes significantly overrepresented in genes significantly downregulated in cKO HSPCs compared to WT HSPCs. b Scatter plot of AUC (representing strength of downregulation) vs log10(FDR), showing the top 25 significantly downregulated genes (red circles) in cKO HSPCs. Scatter plots of AUC vs log10(FDR) highlighting downregulated genes associated with Gene Ontology (GO) biological processes. Red dots found above the dashed line (corresponding to FDR=0.05) represent significantly downregulated genes (FDR<0.05).

Despite the decrease in HPs and HSCs in cKO AGM, NG2-Cre;Runx1fl/fl mice are born with no obvious defects and develop into adulthood. Because of this, we sought to explore the effect of Runx1 deletion in NG2+ PC/vSMC on adult HSPCs. The presence of these progenitors in the adult bone marrow (BM) of mutant mice was analyzed by flow cytometry and compared to WT mice. No significant differences were found in either Lin-Sca1+cKit+ (LSK) (Fig.7a, b) nor LSK CD150+CD48-(SLAM) cell frequencies (Fig.7c, d) between cKO mice and WT controls. We performed HP assays and found that the frequencies of hematopoietic cell colonies were similar in all mutants and WT littermates (Fig.7e, TableS8). To assess the capacity of these cells to reconstitute hematopoiesis in vivo, 5105 bone marrow cells harvested from all genotypes were transplanted into sub-lethally irradiated WT mice recipients. Compared to the control group in which 62.1% (18 out of 29) mice were reconstituted, mice injected with NG2-Cre;Runx1fl/+ or NG2-Cre;Runx1fl/fl BM cells showed a significant reduction in the long-term reconstitution potential, with only 27.3% (3 out of 11, p=0.024) and 20% (4 out of 20, p=0.002) of transplanted mice being reconstituted respectively (Fig.7f, TableS3). In addition, the percentage of donor chimerism was significantly reduced in the cKO group. On average, the donor chimerism with WT cells was 33% compared to the 16% and 9% observed when BM cells from NG2-Cre;Runx1fl/+heterozygous and NG2-Cre;Runx1fl/fl cKO (p=0.002) were injected respectively (Fig.7f, TableS4). The remaining HSCs in the mutant NG2-Cre;Runx1fl/+ and NG2-Cre;Runx1fl/fl adult BM are multilineage, showing similar contributions of donor cells to myeloid or lymphoid cell compartments (Fig.7g), and self-renew (Fig.7h). Interestingly, no NG2+Runx1(GFP)+ cells were detected in adult Runx1-IRES-GFP BM hematopoietic niches (Fig.7i), suggesting that they are exclusive to the embryo and that the BM hematopoietic defect found in adults is developmentally driven.

a, bRepresentative plots and percentages of Lin-Sca1+cKit+ (LSK) and c, dLSK CD150+CD48-(SLAM) bone marrow (BM) cells by flow cytometry of WT/ NG2+/+;Runx1fl/+,NG2+/+;Runx1fl/fl (n=9), HET NG2-Cre;Runx1fl/+ (n=4) and cKO NG2-Cre;Runx1fl/fl (n=4) adult mice is shown. e Colony-forming unit-culture (CFU-C) numbers per 104 adult BM cells; n=WT/HET/cKO: 13/7/8 mice. N=7 independent experiments. Data are meanSEM (TableS8). f Hematopoietic stem cell repopulating potential and donor chimerism of WT and mutant BM cells in vivo. 5105 BM donor WT, HET and cKO cells were injected into 29, 11 and 20 Ly5.1 HET recipients, respectively, with 18, 3 and 4 found to be reconstituted respectively (Table S3, p=0.024 (WT/HET) and p=0.002 (WT/cKO) by Z score test for 2 population proportions). Mice are reconstituted when 5% donor cells are found in the host peripheral blood; p=0.002 (WT/cKO) by Kruskal-Wallis and Dunns post-hoc test (TableS4). g Histograms showing the contribution of CD45.2+CD45.1- donor cells to myeloid cells (CD11b+Gr1+/-), B cells (CD19+) and T cells (CD4/8+) in all reconstituted host mice from (f). (n=WT/HET/cKO=18/3/4), p=0.019 (WT/HET) for B cells by one-way ANOVA and Tukeys post-hoc test. h BM cells from selected reconstituted primary recipients (found in f) were transplanted into multiple irradiated secondary recipients. Mice are reconstituted when 5% donor cells are found in the host peripheral blood (TableS34). i Representative flow cytometric analysis plot of NG2 in Runx1-IRES-GFP adult BM (n=6). All data are presented as Mean values+/-SEM. N=number of independent experiments; n = number of biological samples. Source data for b, d, e, f, g and h are provided as a Source Data file.

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Alabama’s biggest hospital to suspend transfer of embryos after court ruling – ABC News

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U.S. Senators Introduce Bill to Protect Aborted Babies from Medical Experimentation – Daily Citizen

In late January, pro-life senators introduced legislation to protect the human remains of aborted babies from medical experimentation for research purposes.

The bill,S. 3713, is titledProtecting Life and Integrity in Research Act of 2024.

If passed, the measure would prohibit the federal government from funding, approving, or in any way supporting research on aborted babies.

The language of S. 3713 expressly forbids medical research organization to solicit or accept aborted fetal human remains as a research donation.

Senator Cindy Hyde-Smith, chair of theSenate Pro-Life Caucus, is the bills primary sponsor. Seventeen other senators join her ascosponsors.

The law regulating human fetal tissue research is complex. Over the years, federal policy has changed significantly based on the views of each presidential administration.

Currentfederal lawpermits research on fetal human tissue if human embryos are not intentionally created or destroyed for the explicit purpose of research.

According to those statutory stipulations, it is still lawful for taxpayer dollars to fund research on aborted babies if the aborting mothers consent.

Opponents of this practice contend that taxpayers money should not be used to promote unethical research on children.

In apress release, Senator Hyde-Smith called the harvesting and trafficking of aborted babies body parts heinous and unethical.

Proponents tout the possibility of discovering medical advancements by using human fetal tissue, but recentanalysisby the Charlotte Lozier Institute calls that assumption into question.

The reportconcludesthat medical research using ethically non-controversial adult and induced pluripotent stem cells continues to advance in the pursuit of cures and treatments, while embryonic stem cells have largely fallen by the wayside, proving that science does not need to kill in order to cure.

Since 2015, the National Institutes of Healths funding for human fetal tissue research has been as high as $115 million annually. Spending for 2024 isprojectedto be approximately $61 million.

Source: National Institutes of Health

Focus on the Family, asreportedby the Daily Citizen, believes human fetal tissue research requiring the destruction of human beings is a violation of the sanctity of life.

Every human, in every condition from the single cell stage of development to natural death, is made in Gods image and possesses inestimable worth. Our human nature not our size, level of development, environment or functional capacity gives us worth and dignity as human beings. Therefore, devaluing and destroying the life of a human embryo opens the door to the devaluing and destroying any human life.

According to supporters of the legislation, the bill is endorsed by Susan B. Anthony Pro-Life America, Americans United for Life, Catholic Vote, March for Life Action, U.S. Conference of Catholic Bishops Committee on Pro-Life Activities, Students for Life Action, and Concerned Women for America LAC.

A companion measure was introduced in the U.S. House of Representatives last year,H.R. 398, but no action has been taken to advance the legislation.

The Senate bill was referred to the Senate Committee on Health, Education, Labor, and Pensions, where it awaits further action. The Daily Citizen will keep you updated on its progress.

Image from Shutterstock.

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U.S. Senators Introduce Bill to Protect Aborted Babies from Medical Experimentation - Daily Citizen

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