Recent advances in CRISPR-based genome editing technology and … – Military Medical Research

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Recent advances in CRISPR-based genome editing technology and ... - Military Medical Research

Iron & the brain: Where and when neurodevelopmental disabilities … – URMC

Study finds possible cellular origin for impairments associated with gestational iron deficiency

The cells that make up the human brain begin developing long before the physical shape of the brain has formed. This early organizing of a network of cells plays a major role in brain health throughout the course of a lifetime. Numerous studies have found that mothers with low iron levels during pregnancy have a higher risk of giving birth to a child that develops cognitive impairments like autism, attention deficit syndrome, and learning disabilities. However, iron deficiency is still prevalent in pregnant mothers and young children.

The mechanisms by which gestational iron deficiency (GID) contributes to cognitive impairment are not fully understood.The laboratory of Margot Mayer- Proschel, PhD, a professor ofBiomedical GeneticsandNeuroscienceat theUniversity of Rochester Medical Center, was the first todemonstrated that the brains of animals born to iron-deficient mice react abnormally to excitatory brain stimuli, and that iron supplements giving at birth does not restore functional impairment that appears later in life. Most recently, her lab has made a significant progress in the quest to find the cellular origin of the impairment and have identified a new embryonic neuronal progenitor cell target for GID. This study was recently published in the journalDevelopment.

We are very excited by this finding, Mayer-Proschel said, who was awarded a$2 million grant from the National Institute of Child Health & Human Development in 2018to do this work. This could connect gestational iron deficiency to these very complex disorders. Understanding that connection could lead to changes to healthcare recommendations and potential targets for future therapies.

Michael Rudy, PhD, and Garrick Salois, who were both graduate students in the lab and co-first authors of the study, worked backward to make this connection. By looking at the brains of adults and young mice born with known GID, they found disruption of interneurons, cells that control the balance of excitation and inhibition and ensure that the mature brain can respond appropriately to incoming signals. These interneurons are known to develop in a specific region of the embryonic brain called the medial ganglionic eminencewhere specific factors define the fate of early neuronal progenitor cells that then divide, migrate, and mature into neurons that populate the developing cerebral cortex. The researchers found that this specific progenitor cell pool was disrupted in embryonic brains exposed to GID. These findings provide evidence that GID affects the behavior of embryonic progenitor cells causing the creation of a suboptimal network of specialized neurons later in life.

As we looked back, we could identify when the progenitor cells started acting differently in the iron-deficient animals compared to iron normal controls, Mayer-Proschel said. This confirms that the correlation between the cellular change and GID happens in early utero. Translating the timeline to humans would put it in the first three months of gestation before many women know they are pregnant.

Margot Mayer-Proschel

Having identified cellular targets in a mouse model of GID, Neuroscience graduate student Salois in the Mayer-Proschel lab is now establishing a human model of iron deficiency using brain organoidsa mass of cells, in this case that represent a brain. These mini brains that look more like tiny balls that need a microscope to be studied, can be instructed to form specific regions of the ganglionic eminences of the embryonic human brain. With these researchers can mimic the development of the neuronal progenitor cells that are targeted by GID in the mouse.

We believe this model will not only allow us to determine the relevance of our finding in the mouse model for the human system but will also enable us to find new cellular targets for GID that are not even present in mouse models, said Mayer-Proschel. Understanding such cellular targets of this prevalent nutritional deficiency will be imperative to take the steps necessary to make changes to how we think of maternal health. Iron is an important part of that, and the limited impact of iron supplementation after birth makes it necessary to identify alternative approaches,

Additional authors include Janine Cubello, PhD, and Robert Newell at the University of Rochester. This research was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the National Institute of Health, the Toxicology training grant of the Environmental Health Department at the University of Rochester, the New York Stem Cell Training Grant, and the Kilian J. and Caroline F. Schmitt Foundation through the Del Monte Institute for Neuroscience Pilot Program.

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Umbilical Cord Keepsakes Are Going Viral: OB-GYNs Weigh In On … – Scary Mommy

Among the many treasured keepsakes out there to celebrate your little one coming into the world, youre probably aware of the classics: memory books, photo albums, framed hand- and footprints. But if youve been on TikTok lately, you may have noticed a decidedly different kind of keepsake flooding your feed some parents are preserving their umbilical cords as a way to cherish the connection between themselves and their babies, creating a memory they can hold onto for years to come.

If this is the first youre hearing of umbilical cord keepsakes, you likely have some questions. Namely, how does one ask for their umbilical cord? Is it even safe and sanitary to handle it? And how do you transform it into something special, like a heart or a star, especially if youre not particularly crafty or handy?

Granted, if youre being honest, the idea might even make your stomach turn a little especially if youre not a mom whos crunchy enough to have ever considered preserving placental parts for any reason. Thats understandable. But in learning a little more about why certain parents opt for this very intimate art form, you might be surprised to find that you sort of see the appeal. Or, at the very least, you understand why they see the appeal.

The first thing that might pop into your head is, Huh, I didnt even really know people were allowed to keep their umbilical cord. But yep, thats a thing people can (and often) do. Scary Mommy asked two OB-GYNs about the protocol here, and they explained you can certainly ask your obstetrician or someone on your delivery care team to hold onto the umbilical cord. If youre adopting or working with a surrogate, you can also ask the birth mom or gestational carrier for it as well. Most hospitals have policies and processes for patients to request to keep their placentas, which would include the umbilical cord. Its not a very common request, but it does happen, explains Staci Tanouye, MD FACOG.

Still, its not exactly the type of thing you can handle willy-nilly, as caring for an umbilical cord requires some pretty measured safety precautions you will not want to skimp on. Wearing gloves is essential, and Kim Langdon, MD, an OB-GYN with Medzino, explains that parents can request a section of the umbilical cord, placed in a plastic bag, rinsed clear of blood and debris, and refrigerated or have it placed in a desiccator, a specialized sealable container or package that prevents moisture from developing within.

Hospital staff can help ensure that all safety measures are adhered to, with Tanouye adding, As long as its not being ingested and it can be fully dehydrated so it doesnt grow mold, I dont see a safety problem with keeping a portion of the umbilical cord. The biggest precaution is ensuring it is fully dehydrated and dried to minimize the risk of it developing mold.

Bringing it home is only half the adventure, and youre likely also wondering how the heck to transform it into a work of art. Thats where artists like Casey Merrell of uCord Keepsakes can help. Merrell has created umbilical cord keepsakes for parents for more than seven years, working closely with families every step of the way.

Merrell recommends asking someone you trust (dads, doula, birth partners, or another family member) to keep track of the umbilical cord, as things can get chaotic in the hustle and bustle of labor and delivery. She adds that legally, your healthcare provider or birth facility cannot prevent you from taking home the placenta (which includes the umbilical cord), and it is your right to keep it. Depending on the policies of your provider/facility, you may be required to sign a release form or liability form in order to keep your umbilical cord, she adds.

Working with a placenta specialist like Merrell is your best bet for a seamless, stress-free, and safe process: A trained professional is going to be trained/certified in bloodborne pathogens, storage, and sanitation guidelines. Merrell only works with one cord at a time to prevent cross-contamination or any other mix-ups. A professional will also wear all needed PPE and all safety procedures are followed, including supplies sanitized and disposed of between each client, she says. Giving it to a friend or family member might mean theyre dehydrating your cord in a personal kitchen near foodborne items, pets and kids, or other non-trained people, leading to a host of potential mishaps.

Merrell notes that each cord is unique, requiring different preservation methods, and the amount of equipment required often means its cheaper and easier to work with a trained professional. At UCord Keepsakes, it costs $149 total, so by the time you gather a dehydrator, payment paper, finishing/sealing materials, gloves/other PPEs, cutting supplies, sanitation supplies, display box, etc., youll likely be over that price point, she says.

A trained placenta specialist can also create a truly unique work of art, with Merrell noting that some cords are long enough to spell out words like love or the babys name or initials. Metallic gold and rose gold options are most popular for finishing, but some moms like to leave them with a clear coating so you can visibly see the vessels that attach mom/baby.

If youre easily grossed out by bodily fluids and the like, your initial reaction might still be, well, grossed out. But before you brand umbilical cord keepsakes as a ridiculous idea, consider this: It helps some people cope with the grief of losing a child.

Merrell, a mom of four living children and a sleeping little boy, is passionate about providing the service, especially to those who have experienced a stillbirth or infant loss and would like a tangible memory of their angel baby. Most cords do work, but both doctors note some instances in which a parent will not be able to keep the umbilical cord after delivery.

If the cord or placenta is abnormal in any way or was the cause of fetal distress, if there was suspicion of an infection, then you probably wouldn't be allowed to have a section until it had been analyzed by the lab or pathologist, says Langdon, with Tanouye adding that some complications during pregnancy or childbirth (like an infection) might prevent parents from taking it home. In Merrells experience, this is rare, and most parents are able to bring it home without issue.

One final tip, per Langdon: Make sure you collect the cord blood before you cut a section of the cord for future storage as embryonic stem cells, if that is something you plan to do. Otherwise, youll likely get the green light to preserve such this link between you and your little one if thats something that speaks to you. If not, just remember before you bash it... it is very meaningful to some parents.

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Umbilical Cord Keepsakes Are Going Viral: OB-GYNs Weigh In On ... - Scary Mommy

Solnica-Krezel honored for contributions to developmental biology … – Washington University School of Medicine in St. Louis

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Scientist to receive Conklin Medal for work in vertebrate embryonic development

Solnica-Krezel

Lilianna Solnica-Krezel, PhD, the Alan A. and Edith L. Wolff Distinguished Professor and head of the Department of Developmental Biology at Washington University School of Medicine in St. Louis, is to receive the 2023 Edwin G. Conklin Medal from the Society for Developmental Biology. She is being recognized for her significant contributions to the understanding of early embryonic development in vertebrates, with a particular focus on zebrafish as a model organism.

The society awards the Edwin G. Conklin Medal in Developmental Biology annually to recognize developmental biologists who have made extraordinary research contributions to the field and are excellent mentors helping to train the next generation of scientists. Solnica-Krezel will receive the honor in July at the societys annual meeting in Chicago, where she will deliver a lecture.

Studying zebrafish, Solnica-Krezel and her team are focused on understanding the earliest stages of development, when different tissues first arise and are arranged into the body plan. Her team also works with human stem cells to test whether the same processes are relevant in people. The research has implications for understanding miscarriage, birth defects and cancer.

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An old model with new insights: endogenous retroviruses drive the … – Nature.com

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An old model with new insights: endogenous retroviruses drive the ... - Nature.com

How to generate new neurons in the brain – Science Daily

Some areas of the adult brain contain quiescent, or dormant, neural stem cells that can potentially be reactivated to form new neurons. However, the transition from quiescence to proliferation is still poorly understood. A team led by scientists from the Universities of Geneva (UNIGE) and Lausanne (UNIL) has discovered the importance of cell metabolism in this process and identified how to wake up these neural stem cells and reactivate them. Biologists succeeded in increasing the number of new neurons in the brain of adult and even elderly mice. These results, promising for the treatment of neurodegenerative diseases, are to be discovered in the journal Science Advances.

Stem cells have the unique ability to continuously produce copies of themselves and give rise to differentiated cells with more specialized functions. Neural stem cells (NSCs) are responsible for building the brain during embryonic development, generating all the cells of the central nervous system, including neurons.

Neurogenesis capacity decreases with age

Surprisingly, NSCs persist in certain brain regions even after the brain is fully formed and can make new neurons throughout life. This biological phenomenon, called adult neurogenesis, is important for specific functions such as learning and memory processes. However, in the adult brain, these stem cells become more silent or ''dormant'' and reduce their capacity for renewal and differentiation. As a result, neurogenesis decreases significantly with age.The laboratories of Jean-Claude Martinou, Emeritus Professor in the Department of Molecular and Cellular Biology at the UNIGE Faculty of Science, and Marlen Knobloch, Associate Professor in the Department of Biomedical Sciences at the UNIL Faculty of Biology and Medicine, have uncovered a metabolic mechanism by which adult NSCs can emerge from their dormant state and become active.

''We found that mitochondria, the energy-producing organelles within cells, are involved in regulating the level of activation of adult NSCs,'' explains Francesco Petrelli, research fellow at UNIL and co-first author of the study with Valentina Scandella. The mitochondrial pyruvate transporter (MPC), a protein complex discovered eleven years ago in Professor Martinou's group, plays a particular role in this regulation. Its activity influences the metabolic options a cell can use. By knowing the metabolic pathways that distinguish active cells from dormant cells, scientists can wake up dormant cells by modifying their mitochondrial metabolism.

New perspectives

Biologists have blocked MPC activity by using chemical inhibitors or by generating mutant mice for the Mpc1gene. Using these pharmacological and genetic approaches, the scientists were able to activate dormant NSCs and thus generate new neurons in the brains of adult and even aged mice. ''With this work, we show that redirection of metabolic pathways can directly influence the activity state of adult NSCs and consequently the number of new neurons generated,'' summarizes Professor Knobloch, co-lead author of the study. ''These results shed new light on the role of cell metabolism in the regulation of neurogenesis. In the long term, these results could lead to potential treatments for conditions such as depression or neurodegenerative diseases'', concludes Jean-Claude Martinou, co-lead author of the study.

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How to generate new neurons in the brain - Science Daily

This stem cell startup is designing a therapy to restore and boost … – The Boston Globe

That bold vision has now attracted personal investments from several local life science leaders, including George Church, the Harvard University geneticist who has founded and advised dozens of biotech companies. Church told Wang a former postdoctoral researcher in his lab that his thymus cell therapy was one of the most exciting ideas hed come across, since it has the potential to impact almost every person on the planet.

Wangs startup recently raised $7 million in seed financing, bringing the total to $13 million, he said. Thymmunes investors include the biotech venture capital firm Pillar VC and NYBC Ventures, the investment arm of the New York Blood Center. Biotech entrepreneurs and investors Mark Bamforth, James Fordyce, John Maraganore, Judy Pagliuca, Philip Reilly, and Mark de Souza pitched in, too.

Maraganore, the former founding chief executive of Alnylam Pharmaceuticals, a genetic medicines company in Cambridge worth more than $23 billion, said he was fascinated by both the near-term and long-term goals of Wangs vision for what thymus cell therapies might do for rare and common conditions alike.

If successful, it could be pretty transformative, Maraganore said. At the end of the day, we all age and die due to our immune system falling apart, and if theres a way to reconstitute it, that would be pretty cool.

Physicians once thought that the thymus, a small gland situated behind the breastbone, between the lungs, and above the heart, was a dispensable organ. But it plays a vital role in developing immunity.

Its job is to basically be the schoolhouse for T cells, those critical cells in your immune system that help you fight everything from pathogens to cancer, Wang said. From a young age, the thymus also teaches T cells about the inventory of molecules normally found in people so that they dont attack their own body and cause an autoimmune disease.

The thymus is perhaps the most important organ youve never heard of. Many folks dont even know it exists, said Thomas de Vlaam, an investor at Pillar VC. If it works well, its unnoticeable, but once it starts failing for whatever reason, the effects are detrimental.

Thymmunes ambitions span the gamut of thymus biology, from replacing missing thymuses to bolstering shrinking ones, and its technology is largely based on work from a group of scientists at the University of California, San Francisco. In 2010, Audrey Parent, a postdoctoral researcher working with UCSF professors Matthias Hebrok and Mark Anderson, was trying to figure out how to make thymus cells in the lab for the first time.

Parent, now an assistant professor at UCSF, said the project involved a lot of trial and error to find a molecular recipe that could turn a stem cell into a thymus cell. There was no recipe to do that at the time, Parent said. We looked at how the embryo does it, we tried to replicate what nature has been doing really successfully, and then transferred that into a recipe that you can do in a dish.

Their results, published in 2013, used human embryonic stem cells to make thymus cells that were transplanted into mice that lacked a thymus. Crucially, the implants allowed the mice to make their own T cells. Thymmune has licensed patents from UCSF, and like many new startups in the stem cell field, it is forgoing difficult-to-source and ethically fraught embryonic stem cells in favor of induced pluripotent stem cells, or iPSCs, which can be made from adult skin cells.

Sometime in the next few years, Wang plans to start a clinical trial in children who are born without a thymus. Its a ticking time bomb where these kids usually dont survive past one or two years, Wang said.

The Food and Drug Administration approved the first therapy for the condition, called congenital athymia, in 2021. Slices of thymus obtained from organ donations, cultured in a lab and surgically implanted into an infants thigh, improved the chances of surviving the otherwise fatal condition to 76 percent after two years. The results there have been fantastic, Wang said. He hopes to replicate them and make an off-the-shelf product that doesnt require organ donations.

If that approach is successful, Wang wants to use his companys thymus cells as a therapy that helps people getting bone marrow or organ transplants recover more quickly and trains their immune systems to not reject the transplant. He also has plans to develop engineered thymus cells that can quell autoimmune diseases by retraining haywire immune cells to stand down and stop attacking the body.

Wangs ultimate vision, and the one thats especially invigorated investors like Church and Maraganore, is to inject thymus cells into aging people to bolster their immune response. To be clear, theres a lot more we need to do before getting to that point, Wang said. But that is the level of aspiration were aiming for. ... We want to provide everyone the opportunity for healthier aging.

Ryan Cross can be reached at ryan.cross@globe.com. Follow him on Twitter @RLCscienceboss.

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This stem cell startup is designing a therapy to restore and boost ... - The Boston Globe

Enhanced mitochondrial biogenesis promotes neuroprotection in … – Nature.com

Reagents and resources

All the reagents including qPCR primers, antibodies, and software used are listed in Supplementary Table1

Human embryonic stem cell (H7-hESCs; WiCell, Madison, WI, https://www.wicell.org) reporter line with CRISPR-engineered multicistronic BRN3B-P2A-tdTomato-P2A-Thy1.2 construct into the endogenous RGC specific BRN3B locus was used for isogenic control20. CRISPR mutated H7-hESC reporter with OPTNE50K-homozygous mutation (H7-E50K) was done as explained previously25. Patient-derived induced pluripotent stem cells (iPSCs) with E50K mutation24 (iPSC-E50K), E50K mutation corrected to WT by CRISPR in the patient-derived iPSC (iPSC-E50Kcorr)25 with BRN3B::tdTomato reporter were obtained from the Jason Meyer lab. All the above cell lines were grown in mTeSR1 media (mT) at 37C in 5% CO2 incubator on matrigel (MG) coated plates. These cells were maintained by clump passaging using Gentle Cell Dissociation Reagent (GD) after 7080% confluency. Media was aspirated and GD was added to cells followed by incubation at 37C in 5% CO2 incubator for 5min. Next, mT was used to break up the colonies into small clumps by repeated pipetting and then seeded onto new MG plates.

For differentiation, stem cells were dissociated to single cells using accutase for 10min and then quenched with twice the volume of mT with 5M blebbistatin (blebb). The cells were centrifuged at 150xg for 6minutes and resuspended in mT with 5M blebb, then 100,000 cells were seeded per well of a 24-well MG coated plate. The next day, media was replaced with mT without blebb. After 24h, media was replaced with differentiation media (iNS) and further small molecule-based differentiation was carried out with iNS media as elucidated previously23. Differentiation of hRGCswas monitored by tdTomato expression and cells were purified during days 4555 using THY1.2 microbeads and magnetic activated cell sorting system (MACS, Miltenyi Biotec) as explained before20,23. Next, hRGCs were resuspended in iNS media, counted using a hemocytometer and seeded on MG-coated plates, coverslips, or MatTek dishes for experiments.

Purified hRGCs were seeded at 30,000 cells per well of a 96-well MG-coated plate and maintained for 3 days. For measuring mitochondrial mass, hRGCs were labeled with mitochondria-specific MTDR dye. To measure mitochondrial degradation, hRGCs were labeled with 10nM MTDR dye for 1h, washed, and then treated with 10M CCCP for a time course. To measure mitochondrial biogenesis, hRGCs were labeled with MTDR first, treated with 10M CCCP or equal amount of DMSO for 3h, then washed and incubated with fresh media with MTDR for the time course. After treatments, hRGCs were dissociated to single-cell suspension using accutase and analyzed using Attune NxT flow cytometer (Thermo Fisher).

Purified hRGCs were seeded at 500,000 cells per well of 24-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules and time points. DMSO was used as the control as the small molecules were dissolved in DMSO. The cells were lysed and collected in 100l of M-PER extraction buffer with 5mM EDTA and protease inhibitors. Protein quantification was done using a BSA standard following the DC Protein Assay Kit II (Bio-Rad) and measured on microplate reader. Loading samples were prepared with heat denaturation at 95C for 5minutes with laemmli sample buffer (1X). 1020g protein per sample were run on Bio-Rad Mini-PROTEAN TGX precast gels, in running buffer (Tris-Glycine-SDS buffer; Bio-Rad) at 100V until the dye front reached to the bottom. The transfer sandwich was made using PVDF membrane (activated in methanol) in Tris-Glycine transfer buffer (Bio-Rad) with 20% methanol and transferred for 2h at 30V, 4C. For the visualization of proteins, the membranes were blocked in 5% skim milk in TBST (TBS buffer with 20% Tween20) for 2h at room temperature and incubated overnight at 4C in 1:1000 dilution of primary antibodies for PGC1 (Abcam), Phospho-PGC1Ser571 (R&D Systems), PGC1 (Abcam), AMPK (Cell Signaling Technologies, CST), Phospho-AMPKThr172 (CST), LC3B (Sigma), GAPDH (CST), or ACTIN (CST). Membranes were then washed three times for 5minutes each in TBST, followed by 2h of incubation in 5% milk in TBST containing anti-rabbit HRP linked secondary antibody (CST) at 1:10,000 dilution. The membranes were again washed three times with TBST and then placed in Clarity Max Western ECL (Bio-Rad) substrate for 5min. The membranes were imaged in a Bio-Rad ChemiDoc Gel Imager, and the raw integrated density for each band was measured and normalized with respect to the corresponding GAPDH or actin loading control using Image J. Treatment conditions were further normalized to the corresponding DMSO control for each experiment. Complete blots of the representative western blot images, with protein molecular weight marker (Thermo Scientific), are provided in Supplementary Fig.6. Protein bands corresponding to the appropriate size were quantified following product datasheets and published literature.

Purified hRGCs were seeded on MG-coated coverslips (1.5 thickness) at a density of 30,00040,000 cells per coverslip and grown for 3 days. Next, hRGCs were treated with indicated molecules and time points. After treatment the media was aspirated, the cells were washed with 1 PBS, and then fixed with 4% Paraformaldehyde for 15min at 37C. Fixed cells were permeabilized with 0.5% Triton-X100 in PBS for 5min and then washed with washing buffer (1% donkey serum, 0.05% Triton-X100 in PBS) three times for 5min each. Cells were blocked with blocking buffer (5% donkey serum, 0.2% Triton-X100 in PBS) for 1h. After blocking, antibodies against TOM20 (Mouse, Santa Cruz), Tubulin 3 (mouse, Biolegend), RBPMS (rabbit, GeneTex) and Optineurin (Rabbit, Cayman Chemicals) were added (1:200 in blocking buffer) and the coverslips were incubated overnight at 4C. Next, coverslips were washed with washing buffer three times for 5min each and incubated for 2h at room temperature in the dark with fluorophore conjugated anti mouse or rabbit secondary antibodies (1:500). The coverslips were washed with washing buffer three times for 5min each, with 1.43M DAPI added to the second wash. The coverslips were then mounted onto slides with DAKO mounting medium. Visualization of above proteins and nucleus was done by confocal immunofluorescence microscopy using Zeiss LSM700 with 63x/1.4 oil objective. Analysis was carried out using ImageJ with maximum projections of DAPI channel (number of nuclei) and sum projections of TOM20 and OPTN channels for the corresponding confocal z-stacks. For OPTN aggregate size, we analyzed particles from 0.02 a.u. to infinity to account for the small and big aggregates.

Purified hRGCs were seeded at 300,000 cells per well of 24-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules and durations. Media was aspirated and cells were incubated in 200l accutase for 10min and then quenched with 400l iNS media. Cells were centrifuged at 150xg for 6min, media aspirated, and the cell pellets were stored at 20C. RNA was extracted from cell pellets following the kit protocol (Qiagen 74104). The RNA concentration was measured using Nanodrop 2000c (Thermo) and 6l of RNA was used to prepare cDNA following the kit protocol (Abm #G492). Primers were designed as detailed in TableS1 and qPCR were performed using BlasTaq qPCR MasterMix with 100ng total cDNA in a 20l reaction mixture using QuantStudio6 Flex RT PCR system (Applied Biosystems). GAPDH or actin was used as a housekeeping gene in every plate to calculate the Ct values. The Ct was calculated with respect to (w.r.t) the average Ct of the control sample. All conditions were measured by averaging three technical repeats for each biological repeat with total three biological replicates.

Purified hRGCs were seeded at 50,00075,000 cells per well of 96-well MG-coated plates and maintained for 3 days. Cells were then treated with indicated molecules for the designated durations. Media was aspirated and cells were incubated in 30l accutase for 10min and then quenched with 100l iNS media. Cells were centrifuged at 150xg for 6min, media aspirated, and the cell pellets were stored at 20C. DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) and eluted with 30l elution buffer. DNA concentration was measured using Nanodrop 2000c (Thermo). 10ng of DNA was used to measure both mitochondrial ND1 gene and internal control, human nuclear RNase P gene copy numbers, as done previously23.

hRGCs were seeded on MG-coated glass bottom (1.5 thickness)MatTek dishes at 40,000 cells per dish and maintained for 3 days. For the experiments in Fig.2ac, cells were incubated with 250l of JC1 media (1:100 in iNS) for 30min in the incubator, then an additional 1.75ml of dilute JC1 (1:1000 in iNS) was added to the dish. The dish was then transferred to the live cell chamber (5% CO2, 37C, Tokai Hit) and confocal z-stacks were acquired prior to (before) and 10minutes after CCCP (10M) treatment using Zeiss LSM700 with 63x/1.4 oil objective. For experiments in Fig.2df, cells were treated with 10M CCCP or equivalent DMSO for 30min. Next, cells were washed with iNS media, and then incubated with 250l of JC1 media (1:100 in iNS) for 30min in the incubator. The JC1 media was then removed, cells were washed again, and 2ml of new iNS was added to the dish. The dish was then transferred to the live cell chamber and imaged. Analysis was carried out using ImageJ with sum projections of red and green channels. The red fluorescence from the tdTomato expressed by the hRGCs was much less intense than the JC1 red staining of mitochondria, thus the red fluorescence measured from the cytoplasm was considered background and subtracted out of the measurements. For the green fluorescence, background was measured from outside the cell. Red-to-Green ratios were calculated by dividing the red intensity by green intensity. These values were then normalized to the control condition, hRGCWT-before (Fig.2c) or average DMSO ratio for each cell line (Fig.2f).

The 96-well seahorse plate was coated with MG and hRGCs were seeded at 250,000 cells per well and maintained for 2 days. 24h before measurements, media was exchanged with 100l iNS with 1g/ml BX795 or equivalent vehicle control DMSO. A day prior to the assay, the sensor cartridge was fully submerged with 200l of sterile water to hydrate it overnight in a non-CO2, 37C incubator. The next day, sterile water was replaced with pre-warmed XF calibrant buffer(Agilent) and the sensors were again submerged and incubated in the non-CO2, 37C incubator for 4560min. Seahorse media was made by adding stock solutions to XF DMEM to have final concentrations of 21.25mM glucose, 0.36mM sodium pyruvate, and 1.25 mM L-glutamine (Agilent), with pH adjusted to 7.387.42. Depending upon the assay, 20M Oligomycin (Oligo), 20M FCCP, 2.5g/ml Rotenone plus 5M Antimycin A (Rot/AA), and/or 175mM 2-deoxy-d-glucose (2-DG) solutions were then prepared in Seahorse media. In the Seahorse plate with hRGCs, iNS media was carefully exchanged to Seahorse media by removing, 60l iNS from all wells and adding 140l of Seahorse media. Next, 140l of the mixed media was removed by pipette and then an additional 140l seahorse media was added to each well to have a final volume of 180l. 180l of Seahorse media was then added to any empty wells. The plate was placed in Incucyte S3 (Sartorius) and one image of each well was taken for cell area normalization using brightfield and red fluorescence (tdTomato) channels. The hRGC seahorse plate was then placed into a non-CO2, 37C incubator for at least 45min. The reagents were added into their respective ports in the cartridge with the final concentrations in the wells as follows. For ATP rate assay, 2.0M Oligo and 0.25g/ml Rotenone plus 0.5M Antimycin A; for the glycolytic rate assay, 0.25g/ml Rotenone plus 0.5M Antimycin A and 17.5mM 2-DG; and for the Mito stress test, 2.0M Oligo, 2.0M FCCP (optimal concentration from FCCP titration), and 0.25g/ml Rotenone plus 0.5M Antimycin A. After loading all ports, the cartridge was placed into a non-CO2, 37C incubator while the experiment was setup in WAVE software (Agilent). The cartridge was then placed into the XFe96 analyzer (Agilent) and run for calibration. After the machine calibrations were successful, the hRGC seahorse plate was placed into the machine and the assay was run (ATP rate assay, glycolytic rate assay, or Mito stress test). Cell area from Incucyte images were measured using Image J and extrapolated for the total cell area in each well for normalization. The assay results were then exported into the appropriate excel macro using Seahorse Wave Desktop software (Agilent) for analysis.

hRGCs were seeded at 25,000 cells per well of a 96-well clear bottom black-walled plate and maintained for 3 days. The cells were then treated with indicated molecules for the designated time points. The caspase activity was measured using the ApoTox-Glo Triplex assay kit (Promega). 100l of Caspase-Glo 3/7 reagent was added to all wells and incubated for 30minutes at room temperature before measuring luminescence (Caspase). Measurements were normalized to DMSO control.

hRGCs were seeded at 250,000 cells per well on MG-coated 6-well plates and maintained for 3 days. The cells were then treated with 1 g/ml BX795 or equivalent DMSO for 24h. Media was aspirated, 500 l of fixative solution (3% Glutaraldehyde, 0.1M Cacodylate) was added, and the cells were incubated for 15min. Next, hRGCs were scraped and pelleted by centrifugation at 10,000xg for 20minutes. The pellets were fixed overnight at 4C, then rinsed the next day in 0.1M cacodylate buffer, followed by post fixation with 1% osmium tetroxide, 0.1M cacodylate buffer for 1h. After rinsing again with 0.1M cacodylate buffer, the cell pellets were dehydrated through a series of graded ethyl alcohols from 70 to 100%, and 2 changes of 100% acetone. The cell pellets were then infiltrated with a 50:50 mixture of acetone and embedding resin (Embed 812, Electron Microscopy Sciences, Hatfield, PA) for 72h. Specimen vial lids were then removed, and acetone allowed to evaporate off for 3h. Then the pellets were embedded in a fresh change of 100% embedding resin. Following polymerization overnight at 60C the blocks were then ready for sectioning. All procedures were done in centrifuge tubes including the final embedding. Sections with cut at 85nm, placed on copper grids, stained with saturated uranyl acetate, viewed, and imaged on a Tecnai Spirit (ThermoFisher, Hillsboro, OR) equipped with an AMT CCD camera (Advanced Microscopy Techniques, Danvers, MA). 49000X images were analyzed using Image J to measure mitochondrial parameters as explained in Fig.5.

Samples were treated with CCCP or BX795 at different time points as independent biological samples. Statistical tests between two independent datasets were done by Students t-test, each data point within a dataset is from an independent culture wellor cell (Figs.1cf, h, 1m; j, l, 2c, f, 3ad, fi, 4bd, fi, 5b, 6b, e, hl, Supplementary Figs.3b, 4bc). We used t-test rather than ANOVA because we did not want to assume that each group has the same variance. For non-normal data distribution, we performed MannWhitney U test to compare between two independent data sets (Fig.5ce, Supplementary Fig.5b). Graphs were made using GraphPad Prism 9.0 softwareand figures were made using Adobe Illustrator.

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

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Enhanced mitochondrial biogenesis promotes neuroprotection in ... - Nature.com

Will future computers run on human brain cells? Breaking ground on … – Science Daily

A "biocomputer" powered by human brain cells could be developed within our lifetime, according to Johns Hopkins University researchers who expect such technology to exponentially expand the capabilities of modern computing and create novel fields of study.

The team outlines their plan for "organoid intelligence" today in the journal Frontiers in Science.

"Computing and artificial intelligence have been driving the technology revolution but they are reaching a ceiling," said Thomas Hartung, a professor of environmental health sciences at the Johns Hopkins Bloomberg School of Public Health and Whiting School of Engineering who is spearheading the work. "Biocomputing is an enormous effort of compacting computational power and increasing its efficiency to push past our current technological limits."

For nearly two decades scientists have used tiny organoids, lab-grown tissue resembling fully grown organs, to experiment on kidneys, lungs, and other organs without resorting to human or animal testing. More recently Hartung and colleagues at Johns Hopkins have been working with brain organoids, orbs the size of a pen dot with neurons and other features that promise to sustain basic functions like learning and remembering.

"This opens up research on how the human brain works," Hartung said. "Because you can start manipulating the system, doing things you cannot ethically do with human brains."

Hartung began to grow and assemble brain cells into functional organoids in 2012 using cells from human skin samples reprogrammed into an embryonic stem cell-like state. Each organoid contains about 50,000 cells, about the size of a fruit fly's nervous system. He now envisions building a futuristic computer with such brain organoids.

Computers that run on this "biological hardware" could in the next decade begin to alleviate energy-consumption demands of supercomputing that are becoming increasingly unsustainable, Hartung said. Even though computers process calculations involving numbers and data faster than humans, brains are much smarter in making complex logical decisions, like telling a dog from a cat.

"The brain is still unmatched by modern computers," Hartung said. "Frontier, the latest supercomputer in Kentucky, is a $600 million, 6,800-square-feet installation. Only in June of last year, it exceeded for the first time the computational capacity of a single human brain -- but using a million times more energy."

It might take decades before organoid intelligence can power a system as smart as a mouse, Hartung said. But by scaling up production of brain organoids and training them with artificial intelligence, he foresees a future where biocomputers support superior computing speed, processing power, data efficiency, and storage capabilities.

"It will take decades before we achieve the goal of something comparable to any type of computer," Hartung said. "But if we don't start creating funding programs for this, it will be much more difficult."

Organoid intelligence could also revolutionize drug testing research for neurodevelopmental disorders and neurodegeneration, said Lena Smirnova, a Johns Hopkins assistant professor of environmental health and engineering who co-leads the investigations.

"We want to compare brain organoids from typically developed donors versus brain organoids from donors with autism," Smirnova said. "The tools we are developing towards biological computing are the same tools that will allow us to understand changes in neuronal networks specific for autism, without having to use animals or to access patients, so we can understand the underlying mechanisms of why patients have these cognition issues and impairments."

To assess the ethical implications of working with organoid intelligence, a diverse consortium of scientists, bioethicists, and members of the public have been embedded within the team.

Johns Hopkins authors included: Brian S. Caffo, David H. Gracias, Qi Huang, Itzy E. Morales Pantoja, Bohao Tang, Donald J. Zack, Cynthia A. Berlinicke, J. Lomax Boyd, Timothy DHarris, Erik C. Johnson, Jeffrey Kahn, Barton L. Paulhamus, Jesse Plotkin, Alexander S. Szalay, Joshua T. Vogelstein, and Paul F. Worley.

Other authors included: Brett J. Kagan, of Cortical Labs; Alysson R. Muotri, of the University of California San Diego; and Jens C. Schwamborn of University of Luxembourg.

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Will future computers run on human brain cells? Breaking ground on ... - Science Daily

Should we allow genome editing of human embryos? – University of Cambridge news

Andrea Reid-Kelly

I grew up in Leeds with my parents, my older brother and later on my foster brother Scott, who has Downs syndrome. I think growing up with a brother with Downs probably made me a better person. You develop a compassion and understanding for those whose experience is different from your own. This probably has something to do with me going into teaching and Ive taught children with special educational needs since the mid-1990s. I now live in Birmingham with my husband Martin and our daughters, Francesca and Claudia.

I was born with a significant heart condition, which we later learned was caused by Noonan syndrome. This is a genetic condition that can cause heart problems, distinctive facial features, small stature and specific learning difficulties such as dyslexia. But symptoms vary widely and so does its severity some people may never know that they have it. Its estimated that as many as one in 1000 people are born with Noonan syndrome. It can be hereditary or appear by chance, as it did in my case.

I have a couple of the more severe symptoms, I suppose. I had heart surgery when I was four years old and then again when I was 33 years old. My last surgery was three days after my daughters second birthday, which was hard. At four feet nine inches tall, I have proportionate short stature, a form of dwarfism. I also have dyslexia and wonder if I have other specific learning difficulties as well. Although the genetic cause of Noonan is known in many cases, in my case it remains a mystery.

Despite these challenges, I live a happy life and Im proud of what Ive achieved. My condition hasnt stopped me doing anything, except reaching the top shelf in the supermarket! I went through an angry phase when I was younger, but am at peace with who I am and I think dealing with Noonan has given me more confidence and strength to tackle lifes other challenges. I sometimes think my condition is harder on my family than it is on me they cant help worrying about me.

When my husband and I started thinking about having children, it was obviously a really serious decision. There was a 50-50 chance that my children would inherit Noonan syndrome. After a lot of soul searching, I decided that it was worth the risk. But I really pushed my husband to think long and hard about whether he wanted to have children with me. I didnt want him to just say yes because he loved me and hoped for the best.

So far as we know, neither of our daughters have Noonan syndrome. Because the gene that causes my case is unknown, we cant test them to know for sure. I wonder if Francesca has a very mild form, though. There are subtle signs, but then its easy to see what isnt there. Not every trait is necessarily linked to Noonan.

I first met another person with Noonan syndrome some years ago. Theres something powerful about meeting someone with the same genetic condition, you can identify with their experience and bond over challenges. I heard about the citizens jury via Genetic Alliance UK and decided to get involved.

Before the jury, I didnt really know much about editing of human embryos or have a strong opinion about it. Im a bit of a fence-sitter by nature. But by the end of the jury, I went from a neutral opinion to being in favour of parliamentary debate about potentially changing the law to allow editing of human embryos to treat genetic conditions. I felt well-informed and empowered after hearing from experts and others whose experience differed from mine.

One thing I feel very strongly now is that open and honest debate needs to happen about editing of human embryos, because it is incredibly complex. I think about my brother Scott and how the world would be less rich without people like him in it he is so much more than a person with Downs syndrome. We shouldnt automatically seek to remove difference, even if it scares us, because the less we see it the less compassionate we become as a society.

I think personal choice is key. If I could go back in time and had the option, I wouldnt chose to edit my embryos. But I understand that my daughters might want to have that choice, because theres a chance they have Noonan syndrome and could pass that on to their children.

The citizens jury has been an incredibly powerful and inspiring experience, its an amazing tool for debating complex issues. The danger, I think, is that nobody pays attention and that our thoughts have no influence in the end.

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Should we allow genome editing of human embryos? - University of Cambridge news