CRISPR Therapeutics vs Editas Medicine – Securities.io

Gene Editing Hype

Gene editing has for a while been hailed as the new frontier in medicine. The peak enthusiasm with investors on this topic was in early 2020, with the related stocks having cooled off since. No matter the market sentiment, gene editing is still a big deal for medical and pharmaceutical companies as well as patients and doctors.

Gene editing is the next step after gene therapies. Gene therapies add a healthy gene to the genome but leave in place the defective gene. Editing in contrast actually repairs the faulty gene.

Two of the leading firms in the sector are CRISPR Therapeutics and Editas Medicine.

Which one, if any, should you pick as an investment?

Many diseases are due to defective genes, leading to non-functional organs or biochemical processes. They are very often difficult to cure diseases. Infectious diseases can be solved by killing pathogens. Other problems can be solved through surgery or drugs. But when the point of failure is in every cell and requires the body to be changed at the DNA level, this is a lot harder.

For a long time, it was believed that the only solution was gene editing at the early embryo stage, to solve the problem when there is only one cell or at most a few hundred stem cells. And even then, inserting a new, functional gene in defective cells was tricky and prone to failure, as the random entry of the new gene could damage other parts of the genome.

This was until the CRISPR-Cas9 system was discovered. It can be used to target a specific place in the genome. And then to do almost anything molecular biologists want, from knocking-out a gene, entirely deleting it, or also editing it. It can also insert in a controlled fashion entirely new genetic sequences.

This changed everything. Previous methods were too crude to be efficient or safe for most patients. CRISPR brings molecular biology to the next level, allowing precise and in-vivo gene editing to become repeatable and predictable.

Beyond CRISPR-Cas9, researchers have also discovered CRISPR-Cas12. It has slightly different characteristics that might prove better in some cases, like editing multiple genes at once. Or for cell types that do not tolerate Cas-9 well.

While CRISPR Therapeutics favors Cas9, Editas Medicine favors a version of Cas12. If you are technically minded and want to learn more about the difference between the 2 CRISPR systems, I recommend reading this scientific publicationand this article.

The company was founded in 2013 under the name Inception Genomics and went public in 2016.

One of the founders of CRISPR Therapeutics is Emmanuel Charpentier, the discoverer of CRISPR-Cas9 and the Nobel prize of Chemistry in 2020 for that discovery. So it is safe to assume that the company has a crack team when it comes to the scientific side of CRISPR-based gene editing.

Its technology is based on CRISPR-Cas9, allowing for the edition of precisely targeted sections of the genome.

Editas Medicine was founded in 2013 and went public in 2016. It initially started working with Cas9 but is now focused on a proprietary version of Cas12 that they engineered: AsCas12a.

We have covered in detail the unique capacities of Cas-12a in a dedicated article. To resume it shortly:

CRISPR Therapeutics has made the most progress on 2 diseases, Beta-thalassemia and sickle cell diseases (SCD).

This uses an ex-vivo technique: stem cells from the patients are collected, modified/repaired with CRISPR-Cas9, and reintroduced in the body.

Both are under clinical trials in collaboration with Vertex. In June 2022, results from a clinical trial revealedthat 42/44 patients with thalassemia were free from the need for blood transfusion, with the 2 others requiring a lot less blood transfusion.

No serious adverse event was found in SCD patients. Two thalassemia patients had serious adverse events, which have since been healed.

Overall, the blood therapies using CRISPR-Cas9 seem to be a success, and the safety profile acceptable considering how life-threatening and difficult to live with are the diseases treated. You can learn more about the experience of the cured patientin this podcast interviewing one of the participants in the trial.

Another application of CRISPR Therapeutics technology is cancer treatment. The idea is to use modified immune system cells to attack cancer cells. Until now, cells from the patient had to be genetically modified, which took several weeks, which often can be too late for a patients quickly degrading health.

Instead, the company is developing a modified cell that can be manufactured in advance and fit all patients. The method to target the cancer cell is not new, but the possibility to start treatment immediately is. The option to produce a batch of products for hundreds of patients at once is also precious, as it can reduce the complexity and costs of this therapy.

The company has currently 8 candidates in the pipeline, of which 2 already in clinical trials.

CRISPR Therapeutics is also collaborated with the company ViaCyte to improve its product. ViaCyte is aiming to cure type-1 diabetes. This is a disease affecting 8 million peopleand requiring lifelong treatment with insulin.

The issue with ViaCytes current design is that it requires a lifetime of immuno-suppression treatments, which come with their own set of risks and issues. This in turn drastically reduced the size of ViaCytes market.

With the help of CRISPR, ViaCyte is aiming at turning its solution into a lifelong cure for all type-1 diabetes.

Promisingly, the same idea could be used for many other diseases where a specific type of cell needs to be replaced. This could include type-2 diabetes, affecting more than 6% of the worlds population, as well as hepatitis, cirrhosis, or other degenerative diseases.

Each of these 3 applications uses the ex-vivo approach of modifying cells in a lab and re-injecting them in the patients. This is not possible for some diseases, for example, muscular or pulmonary diseases. So CRISPR Therapeutics is also trying to modify the cells of the patients directly in the body, with so-called in-vivo techniques. This either uses viruses as vectors of mRNA techniques not dissimilar to mRNA vaccines.

This is targeting a wide array of diseases including muscular dystrophia and cystic fibrosis (both in partnership with Regeneron), hemophilia (in partnership with Bayer), and cardiac diseases.

In the long run, CRISPR Therapeutics expect the in-vivo technology to become their flagship product and the center of their commercial strategy, able to solve 90% of the most prevalent severe monogenic diseases (see page 35)

Overall, CRISPR therapeutic has done a lot of progress.

It is currently applying for commercialization of its blood therapy products which could concern as many as 30,000 patients in the US and EU. Approval is never a sure thing, but published data last summer of 2022 indicates life-changing efficiency and an acceptable safety profile. Likely, the product could be approved for severe cases at least. This should prove a strong catalyst for the stock as it would be the first product approval for CRISPR Therapeutic.

Further improvement could grow this market to 166,000 patients, or even 450,000 if the in-vivo method proves successful(see the linked presentation page 8).

The cancer treatment trials are still in the early stages, so impossible to predict the outcome. Preliminary data have been encouraging.

The diabetes treatments entered trial on 2ndFebruary 2022. So it is too soon to judge it, but results from this trial could be another strong catalyst for the stock in 2023.

Editas Medicine was previously working, through its EDIT-101 treatment, on curing blindness due to Leber congenital amaurosis 10. The phase 1/2 clinical trial went well, demonstrating the proof of concept.

However, Editas is now looking to license out its technology for this disease, and focus exclusively on its blood disease treatment. It seems the strategic reorientation is due to:

Editas is now focusing on Sickle Cell Disease (SCD), hence going into direct competition with CRISPR Therapeutics own gene editing treatment for SCD.

Editas strategy is counting on the engineered AsCas12a CRISPR system, delivering a superior editing efficiency and specificity than its competitors system using Cas9.

The company is using ASCas12a to activate the genes of ftal hemoglobin in adults, producing functional ftal hemoglobin to replace the one not working in cases of SCD.

The company have also programs at an early stage in oncology (cancer) in partnership with BMS and Immatics. Other organs are also researched, likely for in-vivo therapies. Little has been disclosed about these programs so far.

The initial trial for SCD treatment on 2 patients has shown a good safety profile in the results published on December 2022. The initial results are also demonstrating the proof of concept of the treatment, having increased significantly the hemoglobin levels in the patients blood and reduced or removed symptoms of the disease. Data from additional patients should be published in mid-2023.

The next step is including 40 patients in a clinical trial at phase 1/2, with the first results expected by the end of 2023.

CRISPR Therapeutics valuation in early 2023 has shrunk significantly from a peak of $13.7B in January 2021.

As the company does not have a commercialized product yet, it is reliant on its cash balance and deals with larger pharmaceutical companies.

For example, it register $912M of revenue from its collaboration with Vertex in 2021. This can be compared to $438M in R&D spending and $102M in general administrative spending in the same year. With only 500 employees, the company seems rather lean, efficient, and focused on innovation.

The company has approximately $2B in cash, which should cover the companys needs up to 2024. It has no significant debt or liabilities beyond current operational liabilities and leases for its manufacturing facilities.

Overall, the company finances are sounds, even if it might need to raise more money at one point if its sickle cell disease and thalassemia drugs are not quickly approved. In that respect, the elevated share price of 2021 should have been better utilized to raise funds than risking the current lower valuation.

Like most biotech companies, Editas Medicines valuation is quite lower than its peak at $5.6B in January 2021.

When it comes to the maturity of its portfolio, Editas is just launching now the 40+ patient trials that CRISPR Therapeutics has already finished. So it is likely lagging 1-2 years behind when discussing possible commercialization.

The company has been losing $193M in 2021, of which $142M was spent on R&D. As it currently has $507M in current assets, its liquidity is sufficient for the whole of 2023, even taking into account the extra cost of the incoming clinical trial.

Editas Medicine might need extra funding before reaching commercialization, but this will likely not be the cause of a serious dilution of shareholders, thanks to the solid current cash position. It issued shares worth $203M in 2020 and $249M in 2021, making good use of the then-higher share prices.

Overall, Editas Medicine is at an earlier stage than CRISPR Therapeutics. But thanks to its focused approach centered on only one treatment and disease, it has a similar risk profile when it comes to cash balance and risk of dilution.

CRISPR Therapeutics isthe leader of the sector, benefiting from its first mover advantage, having been founded by the discoverer of Cas9 technology. It also has a much wider portfolio, covering SCD but also another blood disease, cancer, and even diabetes. So its overall potential addressable market is much wider.

It is also more advanced in its clinical trial, having a realistic chance to see at least one product commercialized in a 12-24 months time frame.

Where CRISPR Therapeutics might be lacking, is in its reliance on Cas9 technology, which might be better understood, but slightly less efficient in the long run. It is difficult to judge if these technical differences will result in practical differences in therapeutic efficiency.

Editas Medicine is a trailblazer in turning Cas12a into a practical medical tool. By concentrating its effort on SCD, it is directly targeting CRISPR Therapeutics own SCD treatment. So a lot of the future success or failure of Editas will depend if its treatment for SCD proves superior to CRISPR Therapeutics.

Both company valuations can be considered somewhat equivalent, as CRISPR Therapeutics has a much higher valuation, but also a much more diverse pipeline. Especially as both share a similar risk profile with a large cash cushion enough to cover the next 1-2 years of spending.

It is also possible that both companies will reach commercialization, and share the SCD market on relatively equal terms.

For investors looking at a very innovative and focused company, Editas Medicine might be a favored choice.

For investors looking at a more spread R&D risk, CRISPR Therapeutics wider pipeline should prove more reassuring. The upside in the 4-6 years timeframe of CRISPR Therapeutics might be also larger, thanks to its venture into the very large diabetes market.

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CRISPR Therapeutics vs Editas Medicine - Securities.io

Embryonic Stem Cells – The Definitive Guide | Biology Dictionary

Adult stem cells maintain and repair tissues throughout the body

Embryonic stem cells are pluripotent cells derived from a 3 5 day old human embryo. They have the unique potential to develop into any of the other 200+ human cell types, and can significantly further our understanding of human development and diseases.

Embryonic stem cells also have important applications in drug development, and may one day be used to treat currently incurable conditions.

Stem cells are cells that have the potential to differentiate and give rise to other types of body cells. They are the basic materials from which all of the bodys specialized cells are made during whole-body development and, in adulthood, are used to maintain and repair body tissues. There are two types of human stem cells, and these are embryonic stem cells and adult stem cells.

Embryonic stem cells (ESCs) are stem cells derived from a 3 5 day old human embryo (AKA a blastocyst). ESCs are pluripotent, meaning they have the potential to become any of the other 200+ types of cells found in the human body. As the embryo develops, ESCs divide and differentiate to form the full complement of human body cells required for healthy function.

The first differentiation event in human embryos begins around 5 days after fertilization, so ESCs must be harvested before this time if they are to be used in medicine and research. At this early developmental stage, the cells of the embryo form an undifferentiated mass and have not yet taken on the characteristics or functions of specialized adult cells.

The ability of ESCs to develop into all other types of human cells makes them an invaluable research tool. Studies involving ESCs can advance our understanding of human development, disease treatment, and drug efficacy.

ESCs can be grown (or cultured) in a laboratory. When kept under the right conditions, stem cells will grow and divide indefinitely, without becoming differentiated. However, they will still maintain their ability to differentiate, making the ESC culture a convenient and renewable reservoir of human cells. When used in research, ECSs are converted into their desired cell types by manipulating the culture conditions.

Scientists can use stem cells to further their understanding of human development and diseases. By studying embryonic stem cells, researchers hope to learn how they differentiate to form tissues and organs, how diseases and conditions develop in these tissues, and how age affects their function.

Scientists can also use ESCs to test and develop new drugs and to help them identify new potential treatments for diseases like Parkinsons disease, heart failure, and spinal cord injuries.

ESCs have enormous potential in the development of restorative or regenerative medicine, in which damaged tissues are replaced by healthy ones. Currently, several stem cell therapies are possible and could be used to treat a variety of injuries and diseases. These include spinal cord injuries, retinal and macular degeneration, heart failure, type 1 diabetes, and tendon rupture.

However, research into the use of ESCs for regenerative medicine are ongoing, and better understanding is required before modern medicine can harness their full potential. In the future, scientists hope that stem cell therapies can be used to treat currently incurable or difficult to treat conditions, such as AIDS or certain types of cancer.

Currently, the most common stem cell therapy is multipotent hematopoietic stem cell (HSC) transplantation. This treatment involves the transplantation of hematopoietic (or blood) stem cells and is usually used to treat diseases affecting the blood cells, such as leukemia and anemia.

ESCs can also be used in the development of new drugs, which must be tested on living tissues to determine their efficacy and any possible side effects.

Stem cells cultured in the laboratory can be stimulated to differentiate into any type of human tissue, so they are commonly used in preclinical drug trials. Once the potential and risks of the new drug have been determined using stem cells, the treatment can be used in animal tests and, eventually, human clinical trials.

The discovery of ESCs has led to numerous breakthroughs in the field of medical research, and their potential as the basis for new therapies and drugs is enormous. However, there is ethical controversy surrounding the use of ESCs in research, primarily because harvesting these cells involves destroying a human embryo.

For those who believe that life begins at conception, this raises moral objections. Opponents of stem cell research believe that embryos have the same rights as any other human beings, and shouldnt be disposed of in the name of science.

Those who support the use of ESCs in medical research may argue that the embryos do not yet qualify as humans, as they are destroyed in the very early stages of development. ESCs are harvested at around day 5 of development when the embryo (or blastocyst) is nothing more than a mass of undifferentiated cells.

Embryos used as a source of ESCs are frequently obtained from IVF clinics, where they have been frozen following fertilization. Guidelines created by the National Institute of Health state that embryos can only be used for this purpose when they are no longer needed (meaning they will never be implanted in a womans uterus). Such embryos would eventually be discarded anyway, so it can be argued that they would be better used to advance medical research.

Adult stem cells (AKA somatic stem cells) are stem cells that are found in most adult tissues.

They can develop into other types of cells but, unlike, ESCs, they are not pluripotent (able to develop into any other type of cell). Adult stem cells are either multipotent (able to develop into a limited number of closely related cells) or unipotent (able to develop into just one type of cell).

Their main function is to maintain and repair the tissue in which they are found and to replace cells that die as a result of injury or disease.

Mesenchymal stem cells are found in many adult tissues, including the umbilical cord, bone marrow, and fat tissue. In the bone marrow, mesenchymal stem cells differentiate to form bone, cartilage, and fat cells.

Neural stem cells are found in the brain and develop into nerve cells and their supporting cells (glial cells).

Hematopoietic stem cells are found in the bone marrow and peripheral blood. They give rise to all kinds of blood cells, including red blood cells, white blood cells, and platelets.

Skin stem cells are found in the basal layer of the epidermis and form keratinocytes for the continuous regeneration of the epidermal layers.

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Embryonic Stem Cells - The Definitive Guide | Biology Dictionary

Pluripotent embryonic stem cells and multipotent adult germline stem …

Title & authors Abstract Similar articles Cited by Publication types MeSH terms Related information LinkOut - more resources . 2010 Nov;16(11):846-55. doi: 10.1093/molehr/gaq060. Epub 2010 Jul 12.

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DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs).

Zovoilis A, Pantazi A, Smorag L, Opitz L, Riester GS, Wolf M, Zechner U, Holubowska A, Stewart CL, Engel W. Zovoilis A, et al. Mol Hum Reprod. 2010 Nov;16(11):793-803. doi: 10.1093/molehr/gaq053. Epub 2010 Jun 21. Mol Hum Reprod. 2010. PMID: 20566704

Zovoilis A, Nolte J, Drusenheimer N, Zechner U, Hada H, Guan K, Hasenfuss G, Nayernia K, Engel W. Zovoilis A, et al. Mol Hum Reprod. 2008 Sep;14(9):521-9. doi: 10.1093/molehr/gan044. Epub 2008 Aug 12. Mol Hum Reprod. 2008. PMID: 18697907

Nolte J, Michelmann HW, Wolf M, Wulf G, Nayernia K, Meinhardt A, Zechner U, Engel W. Nolte J, et al. Differentiation. 2010 Nov-Dec;80(4-5):184-94. doi: 10.1016/j.diff.2010.08.001. Epub 2010 Sep 1. Differentiation. 2010. PMID: 20810205

Kerr CL, Shamblott MJ, Gearhart JD. Kerr CL, et al. Methods Enzymol. 2006;419:400-26. doi: 10.1016/S0076-6879(06)19016-3. Methods Enzymol. 2006. PMID: 17141064 Review.

Petitte JN, Liu G, Yang Z. Petitte JN, et al. Mech Dev. 2004 Sep;121(9):1159-68. doi: 10.1016/j.mod.2004.05.003. Mech Dev. 2004. PMID: 15296979 Review.

Han Z, Zhang Q, Zhu Y, Chen J, Li W. Han Z, et al. Stem Cells Int. 2020 Jul 6;2020:8863539. doi: 10.1155/2020/8863539. eCollection 2020. Stem Cells Int. 2020. PMID: 32695182 Free PMC article. Review.

Kalasauskas D, Sorokin M, Sprang B, Elmasri A, Viehweg S, Salinas G, Opitz L, Rave-Fraenk M, Schulz-Schaeffer W, Kantelhardt SR, Giese A, Buzdin A, Kim EL. Kalasauskas D, et al. Cancers (Basel). 2020 Mar 1;12(3):570. doi: 10.3390/cancers12030570. Cancers (Basel). 2020. PMID: 32121554 Free PMC article.

Bai Y, Zhu C, Feng M, Wei H, Li L, Tian X, Zhao Z, Liu S, Ma N, Zhang X, Shi R, Fu C, Wu Z, Zhang S. Bai Y, et al. Stem Cell Res Ther. 2018 Jul 18;9(1):200. doi: 10.1186/s13287-018-0931-0. Stem Cell Res Ther. 2018. PMID: 30021628 Free PMC article.

Imamura M, Lin ZY, Okano H. Imamura M, et al. Reprod Med Biol. 2012 Jun 19;12(1):1-14. doi: 10.1007/s12522-012-0131-z. eCollection 2013 Jan. Reprod Med Biol. 2012. PMID: 29699125 Free PMC article. Review.

Saeidi S, Shapouri F, de Iongh RU, Casagranda F, Sutherland JM, Western PS, McLaughlin EA, Familari M, Hime GR. Saeidi S, et al. PLoS One. 2018 Jan 11;13(1):e0190925. doi: 10.1371/journal.pone.0190925. eCollection 2018. PLoS One. 2018. PMID: 29324788 Free PMC article.

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for Human Stem Cell Research – ahrq.gov

NICHD ADMINISTRATIVE SUPPLEMENTS FOR HUMAN EMBRYONIC STEM CELL RESEARCH RELEASE DATE: January 24, 2003 NOTICE: NOT-HD-03-005Update: July 7, 2009 This Notice is superseded by NIH-OD-09-116 NIH Guidelinesfor Human Stem Cell ResearchNational Institute of Child Health and Human Development (NICHD) (http://www.nichd.nih.gov/)The National Institute of Child Health and Human Development (NICHD) announces the availability of administrative supplements to NICHD grantees to conduct research using human embryonic stem cell lines (ESCs) in accordance with the NIH-wide announcement and guidancethat can be found athttps://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-006.html. The human ESCs to be used must be listed on the NIH Human Embryonic Stem Cell Registry (http://escr.nih.gov/). Principal Investigators of NICHD-funded R01, R37, and P01 grants may request an administrative supplement not to exceed $75,000 direct costs (three modules) per year for two years. There must be at least two years of funding remaining on the parent grant at the time the supplement is awarded. It is intended that awards will be initiated in FY 2003 and FY 2004. Requests must be submitted not later than July 1, 2003 for FY 2003 funding or July 1, 2004 for FY 2004 funding.The work proposed must be within the scope of the parent R01, R37 or P01 grant. For example, investigators may apply concepts and technologies being used on any nonhuman adult or embryonic cells in the funded project to the study of human ESCs. The proposed research can utilize the full range of cell biological, genetic or molecular approaches. The request for an administrative supplement must include a careful description of the work proposed, an explanation of the relationship to the parent grant, and a justification for the study. The NICHD intends to commit up to $500,000 direct costs per year for this initiative in FY 2003 and FY 2004. This is a one-time announcement. However, the NICHD may re-release the announcement depending upon the needs of the NICHD scientific community and the availability of funds. Requests will be reviewed by NICHD staff. Awards will be dependent upon the receipt of qualified requests and the availability of funds. Grantees may request funds for small items of equipment, supplies, purchase of human ESCs, and personnel to work with human ESCs. Funds may also be requested to support travel and other costs needed to acquire necessary expertise in the handling of human ESCs. Investigators must independently contact the human ESC providers listed on the NIH Registry and make arrangements to obtain the cell lines, including any required material transfer agreements (MTA). Applicants must indicate which human ESC lines will be used. A letter indicating that the provider has agreed to supply the human ESC line must be furnished either with the application (see below) or just prior to the award. The investigators also should either demonstrate prior ability to work with human ESCs or outline plans for obtaining training to culture human ESCs. The human ESC providers, other laboratories with ESC experience, or laboratory training courses on ESC methods are potential means of obtaining this training. Other means for acquiring human ESC expertise may be proposed. Application ProceduresIn order to apply for an administrative supplement, it is advisable to first discuss your request with the NICHD Program Director who manages your grant or with Dr. Tasca at the address below. After this discussion, send an original letter, co-signed by the business official of the grantee institution, to your Program Director, with one copy to Dr. Tasca (below) and one copy to Ms. Hancock (below). The letter (two page limit) should include: 1) an abstract of the proposed supplemental activity and how it is related to the parent grant; 2) a description of how the requested supplement will provide the resources and expertise necessary to design and perform the experiments using human ESCs; 3) the NIH code for the selected human ESC line(s); 4) details of the budget items requested and funding period; and 5) current contact information for the Principal Investigator, including postal and email addresses. Although these descriptions should be as concise as possible, sufficient detail must be provided to allow the NICHD to determine if the request qualifies as an administrative supplement. INQUIRIESDirect inquiries regarding program and scientific issues to: Dr. Richard J. Tasca Reproductive Sciences Branch Center for Population Research National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8B01, MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 435-6973 FAX: (301) 480-2389 Email: rt34g@nih.gov Direct questions about financial or grants management issues to: Kathy Hancock Grants Management Branch National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8A17, MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 496-5482 FAX: (301) 480-4782 Email: kh47d@nih.gov

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Single mutation helped separate human, Neanderthal brains – Big Think

How the modern human brain evolved, and how it differs from the brains of Neanderthals and other extinct hominin species, is an open question. Expansion of the neocortex was a key event in human brain evolution, and now researchers in Germany say they have identified a genetic mutation that drove this process.

Anneline Pinson of the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden and her colleagues examined a gene called TKTL1, which is active in immature neurons in the fetal neocortex, and is also implicated in the proliferation of brain tumor cells.

TKTL1 encodes an enzyme consisting of 596 amino acid residues and is one of just a few genes whose DNA sequence differs between humans and extinct archaic hominins. In the Neanderthal genome, residue 317 is the amino acid lysine, but in humans, this has been substituted with arginine. Such seemingly tiny differences can matter greatly.

Pinson and her colleagues analyzed previously published human fetal transcriptome datasets, revealing TKTL1 is expressed in a specific population of neural stem cells in the developing nervous system from nine weeks of gestation onward, and that its levels then increase in the immature frontal lobe, but not other areas. The human fetal TKTL1 protein is a shorter form, however, containing only 540 residues, with the aforementioned substitution at position 261.

TKTL1 is not expressed in the embryonic mouse cortex, but when the researchers inserted the human gene into mouse embryos, it increased the number of stem cells that give rise to frontal cortex neurons, resulting in more newborn neurons at later stages of development. Insertion of the Neanderthal TKTL1 gene had no such effect.

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The researchers also inserted human TKTL1 into ferret embryos, which normally express the Neanderthal-like, lysine-containing variant of the gene. This, too, increased the number of neural progenitors and newborn neurons, leading to an expansion of the upper layers of the neocortex.

Conversely, deleting TKTL1 from human fetal brain tissue reduced the number of neural stem cells, and inserting the Neanderthal variant into lab-grown cerebral organoids reduced the number of stem cells and neurons derived from them.

Finally, Pinson and her colleagues determined the function of the TKTL1 enzyme. Their experiments showed that it promotes the synthesis of fatty acids that are inserted into the neural stem cell membrane, which are crucial for the outgrowth of their fibers and their proliferation.

These findings show that a mutation in the DNA sequence of the TKTL1 gene, causing a single amino acid substitution in the human protein sequence, is responsible for the observed effects on neural stem cell behavior. The researchers conclude that this simple genomic change may contribute to the differences in size and shape of the human and Neanderthal neocortex.

Brain development is an extremely complex process, however, and it is highly unlikely that a single genetic event was responsible for evolution of the human brain. Indeed, the process of human brain evolution likely involved many hundreds of genes and multiple different types of genetic events, including changes in non-coding DNA sequences, gene deletions and duplications, jumping genes, and other large-scale genomic changes.

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Single mutation helped separate human, Neanderthal brains - Big Think

Regenerative Medicine Global Market to Surpass $40.7 Billion by 2030 at a CAGR of 12.75% – PR Newswire

DUBLIN, Sept. 7, 2022 /PRNewswire/ --The "Regenerative Medicine Global Market Opportunities And Strategies To 2031" report has been added to ResearchAndMarkets.com's offering.

This report describes and explains the global regenerative medicine market and covers 2016 to 2021, termed the historic period, and 2021 to 2026 termed the forecast period, along with further forecasts for the period 2026-2031. The report evaluates the market across each region and for the major economies within each region.

The global regenerative medicine market reached a value of nearly $7,282.2 million in 2020, having increased at a compound annual growth rate (CAGR) of 54.1% since 2015. The market is expected to grow from $7,282.2 million in 2020 to $22,373.7 million in 2025 at a rate of 25.2%. The market is then expected to grow at a CAGR of 12.7% from 2025 and reach $40,710.1 million in 2030.

Growth in the historic period in the regenerative medicine market resulted from rising prevalence of chronic diseases, emerging markets growth, implementation of the 21st century cures act, rapid growth in aging population, and the improvement in healthcare awareness and expenditure. The market was restrained by high cost of cell and gene therapies, ethical concerns related to the use of embryonic stem cells in research and development, and inadequate reimbursements.

Going forward, rising demand for organ transplantations, growth in healthcare expenditure, technological advancements in regenerative medicines, rising investments in regenerative medicine research, and changes in lifestyles. Factors that could hinder the growth of the market in the future include rising popularity of alternative therapies and natural remedies, low per capita healthcare expenditure, and tissue-engineered products and biomaterials are lagging in adoption.

The regenerative medicine market is also segmented by end-use into ambulatory surgical centers, hospitals and clinics, and others. The hospitals and clinics segment was the largest segment of the regenerative medicine market segmented by end-use, accounting for 63.8% of the total in 202o. Going forward, hospitals and clinics segment is expected to be the fastest growing segment in the regenerative medicine market segmented by end-use, at a CAGR of 25.3% during 2020-2025.

The regenerative medicine market is also segmented by application into musculoskeletal, oncology, dental, wound care and others. The oncology segment was the largest segment of the regenerative medicine market segmented by application, accounting for 60.0% of the total in 2020. Going forward, musculoskeletal segment is expected to be the fastest growing segment in the regenerative medicine market segmented by application, at a CAGR of 27.4% during 2020-2025.

North America was the largest region in the regenerative medicine market, accounting for 53.3% of the total in 2020. It was followed by the Western Europe, Asia Pacific, and then the other regions. Going forward, the fastest-growing regions in the regenerative medicine market will be Middle East and South America where growth will be at CAGRs of 72.4% and 71.9% respectively during 2020-2025. These will be followed by Eastern Europe and Asia Pacific, where the markets are expected to register CAGRs of 57.8% and 49.7% respectively during 2020-2025.

Market Trends And Strategies

Markets Covered:

Key Topics Covered:

1. Regenerative Medicine Market Executive Summary

2. Table of Contents

3. List of Figures

4. List of Tables

5. Report Structure

6. Introduction

7. Regenerative Medicine Market Characteristics

8. Regenerative Medicine Market Trends And Strategies

9. Impact Of COVID-19 On Regenerative Medicine

10. Global Regenerative Medicine Market Size And Growth

11. Global Regenerative Medicine Market Segmentation

12. Regenerative Medicine Market, Regional And Country Analysis

13. Asia-Pacific Regenerative Medicine Market

14. Western Europe Regenerative Medicine Market

15. Eastern Europe Regenerative Medicine Market

16. North America Regenerative Medicine Market

17. South America Regenerative Medicine Market

18. Middle East Regenerative Medicine Market

19. Africa Regenerative Medicine Market

20. Regenerative Medicine Global Market Competitive Landscape

21. Key Mergers And Acquisitions In The Regenerative Medicine Market

22. Regenerative Medicine Market Opportunities And Strategies

23. Regenerative Medicine Market, Conclusions And Recommendations

24. Appendix

Companies Mentioned

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Regenerative Medicine Global Market to Surpass $40.7 Billion by 2030 at a CAGR of 12.75% - PR Newswire

Scientists use stem cells to create synthetic mouse embryos – ABC News

Scientists have created synthetic mouse embryos from stem cells without a dad's sperm or a mom's egg and womb

ByLAURA UNGAR AP Science Writer

Scientists have created synthetic mouse embryos from stem cells without a dad's sperm or a mom's egg or womb.

The lab-created embryos mirror a natural mouse embryo up to 8 days after fertilization, containing the same structures, including one like a beating heart.

In the near term, researchers hope to use these so-called embryoids to better understand early stages of development and study mechanisms behind disease without the need for as many lab animals. The feat could also lay the foundation for creating synthetic human embryos for research in the future.

We are undoubtedly facing a new technological revolution, still very inefficient but with enormous potential, said Llus Montoliu, a research professor at the National Biotechnology Centre in Spain who is not part of the research. It is reminiscent of such spectacular scientific advances as the birth of Dolly the sheep and others.

A study published Thursday in the journal Nature, by Magdalena Zernicka-Goetz at the California Institute of Technology and her colleagues, was the latest to describe the synthetic mouse embryos. A similar study, by Jacob Hanna at the Weizmann Institute of Science in Israel and his colleagues, was published earlier this month in the journal Cell. Hanna was also a coauthor on the Nature paper.

Zernicka-Goetz, an expert in stem cell biology, said one reason to study the early stages of development is to get more insight into why the majority of human pregnancies are lost at an early stage and embryos created for in vitro fertilization fail to implant and develop in up to 70% of cases. Studying natural development is difficult for many reasons, she said, including the fact that very few human embryos are donated for research and scientists face ethical constraints.

Building embryo models is an alternative way to study these issues.

To create the synthetic embryos, or embryoids, described in the Nature paper, scientists combined embryonic stem cells and two other types of stem cells all from mice. They did this in the lab, using a particular type of dish that allowed the three types of cells to come together. While the embryoids they created werent all perfect, Zernicka-Goetz said, the best ones were indistinguishable from natural mouse embryos. Besides the heart-like structure, they also develop head-like structures.

This is really the first model that allows you to study brain development in the context of the whole developing mouse embryo, she said.

The roots of this work go back decades, and both Zernicka-Goetz and Hanna said their groups were working on this line of research for many years. Zernicka-Goetz said her group submitted its study to Nature in November.

Scientists said next steps include trying to coax the synthetic mouse embryos to develop past 8 days with the eventual goal of getting them to term, which is 20 days for a mouse.

At this point, they struggle to go past the 8 1/2-day mark, said Gianluca Amadei, a coauthor on the Nature paper based at the University of Cambridge. We think that we will be able to get them over the hump, so to speak, so they can continue developing.

The scientists expect that after about 11 days of development the embryo will fail without a placenta, but they hope researchers can someday also find a way to create a synthetic placenta. At this point, they don't know if they will be able to get the synthetic embryos all the way to term without a mouse womb.

Researchers said they dont see creating human versions of these synthetic embryos soon but do see it happening in time. Hanna called it the next obvious thing.

Other scientists have already used human stem cells to create a blastoid, a structure mimicking a pre-embryo, that can serve as a research alternative to a real one.

Such work is subject to ethical concerns. For decades, a 14-day rule on growing human embryos in the lab has guided researchers. Last year, the International Society for Stem Cell Research recommended relaxing the rule under limited circumstances.

Scientists stress that growing a baby from a synthetic human embryo is neither possible nor under consideration.

Perspective on this report is important since, without it, the headline that a mammalian embryo has been built in vitro can lead to the thought that the same can be done with humans soon, said developmental biologist Alfonso Martinez Arias of the Universitat Pompeu Fabra in Spain, whose group has developed alternative stem cell based models of animal development.

"In the future, similar experiments will be done with human cells and that, at some point, will yield similar results," he said. "This should encourage considerations of the ethics and societal impact of these experiments before they happen.

The Associated Press Health and Science Department receives support from the Howard Hughes Medical Institutes Department of Science Education. The AP is solely responsible for all content.

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Scientists use stem cells to create synthetic mouse embryos - ABC News

See-through zebrafish, new imaging method put blood stem cells in high-resolution spotlight – University of Wisconsin-Madison

Tracing features in a large 3D electron microscopy dataset reveals a zebrafish blood stem cell (in green) and its surrounding niche support cells, a group photo method that will help researchers understand factors that contribute to blood stem cell health which could in turn help develop therapies for blood diseases and cancers. Image by Keunyoung Kim.

MADISON For the first time, researchers can get a high-resolution view of single blood stem cells thanks to a little help from microscopy and zebrafish.

Researchers at the University of WisconsinMadison and the University of California San Diego have developed a method for scientists to track a single blood stem cell in a live organism and then describe the ultrastructure, or architecture, of that same cell using electron microscopy. This new technique will aid researchers as they develop therapies for blood diseases and cancers.

Currently, we look at stem cells in tissues with a limited number of markers and at low resolution, but we are missing so much information, says Owen Tamplin, an assistant professor in UWMadisons Department of Cell & Regenerative Biology, a member of the Stem Cell & Regenerative Medicine Center, and a co-author on the new study, which was published Aug. 9 in eLife. Using our new techniques, we can now see not only the stem cell, but also all the surrounding niche cells that are in contact.

The niche is a microenvironment found within tissues like the bone marrow that contain the blood stem cells that support the blood system. The niche is where specialized interactions between blood stem cells and their neighboring cells occur every second, but these interactions are hard to track and not clearly understood.

As a part of the new study, Tamplin and his co-lead author, Mark Ellisman, a professor of neuroscience at UC San Diego, identified a way to integrate multiple types of microscopic imaging to investigate a cells niche. With the newly developed technique that uses confocal microscopy, X-ray microscopy, and serial block-face scanningelectron microscopy, researchers will now be able to track the once elusive cell-cell interactions occurring in this space.

This has allowed us to identify cell types in the microenvironment that we didnt even know interacted with stem cells, which is opening new research directions, Tamplin says.

As a part of this study, Tamplin, and his colleagues, including co-first authors Sobhika Agarwala and Keunyoung Kim, identified dopamine beta-hydroxylase positive ganglia cells, which were previously an uncharacterized cell type in the blood stem cell niche. This is crucial, as understanding the role of neurotransmitters like dopamine in regulating blood stem cells could lead to improved therapeutics.

Transplanted blood stem cells are used as a curative therapy for many blood diseases and cancers, but blood stem cells are very rare and difficult to locate in a living organism, Tamplin says. That makes it very challenging to characterize them and understand how they interact and connect with neighboring cells.

While blood stem cells are difficult to locate in most living organisms, the zebrafish larva, which is transparent, offers researchers a unique opportunity to view the inner workings of the blood stem cell niche more easily.

Thats the really nice thing about the zebrafish and being able to image the cells, Tamplin says of animals transparent quality. In mammals, blood stem cells develop in utero in the bone marrow, which makes it basically impossible to see those events happening in real time. But, with zebrafish you can actually watch the stem cell arrive through circulation, find the niche, attach to it, and then go in and lodge there.

While the zebrafish larva makes it easier to see blood stem cell development, specialized imaging is needed to find such small cells and then detail their ultrastructure. Tamplin and his colleagues spent over six years perfecting these imaging techniques. This allowed them to see and track the real-time development of a blood stem cell in the microenvironment of a live organism, then zoom in even further on the same cell using electron microscopy.

First, we identified single fluorescently labeledstem cells bylight sheet or confocal microscopy, Tamplin says. Next, we processed the same sample forserial block-face scanningelectron microscopy. We then aligned the 3D light and electron microscopy datasets. Byintersecting these different imaging techniques,we could see the ultrastructure of single rare cells deep inside a tissue. This also allowed us to find all the surrounding niche cellsthat contact a blood stem cell. We believe our approach will be broadly applicable for correlative light and electron microscopy in many systems.

Tamplin hopes that this approach can be used for many other types of stem cells, such as those in the gut, lung, and the tumor microenvironment, where rare cells need to be characterized at nanometer resolution. But, as a developmental biologist, Tamplin is especially excited to see how this work can improve researchers understanding of how the blood stem cell microenvironment forms.

I think this is really exciting because we generate all of our blood stem cells during embryonic development, and depending on what organism you are, a few hundred or maybe a few thousand of these stem cells will end up producing hundreds of billions of new blood cells every day throughout your life, Tamplin says. But we really dont know much about how stem cells first find their home in the niche where theyre going to be for the rest of the life of the organism. This research will really help us to understand how stem cells behave and function. A better understanding of stem cell behavior, and regulation by surrounding niche cells, could lead to improved stem cell-based therapies.

This research was supported by grants from the National Institutes of Health (R01HL142998, K01DK103908, 1U24NS120055-01, R24 GM137200) and the American Heart Association (19POST34380221).

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See-through zebrafish, new imaging method put blood stem cells in high-resolution spotlight - University of Wisconsin-Madison

First-Ever Embryos With Monkey And Human Cells; Scientists Divided Over The Creation – Boldsky

Wellness

oi-Amritha K

For the first time, scientists have successfully grown monkey embryos containing human cells, marking a milestone in a rapidly advancing field that has raised ethical concerns.

Human stem cells may one day be used to grow new organs for people with failing hearts or kidneys by slipping them into the embryos of other animals. To achieve this goal, researchers have created the first embryos that contain human and monkey cells.

Researchers may be able to refine techniques for growing human tissue in species better suited for transplantation, such as pigs, by studying these chimaeras [1].

In genetics, a chimaera is an organism or tissue that contains at least two sets of DNA, most commonly resulting from the fusion of several zygotes (fertilized eggs). In Greek mythology, the term Chimera refers to a fire-breathing monster that was part lion, part goat, and part dragon [2].

According to the study published in the journal Cell, researchers in the United States and China injected 25 pluripotent stem cells (pluripotent stem cells of the body are capable of self-renewal and give rise to all cells of the body's tissues) from humans into monkey embryos.

After one day, researchers detected the development of human cells in 132 embryos, and the embryos ultimately survived for 19 days.

"The paper is a landmark in the stem cell and interspecies chimaera fields. The findings hint at mechanisms by which cells of one species can adjust to survive in the embryo of another", added experts [3].

According to the researchers, a third of the chimaeras had human cells present after 13 days. There appears to be an integration of human cells with monkey cells, and the human cells have begun to specialize into different types of cells that will develop into different organ types. A few previous studies by researchers in the same study group had explored embryo development along the same lines:

Researchers identified molecular pathways that were turned on or up in the chimaeras, possibly promoting the integration of human and monkey cells. Researchers believe that manipulating some of these pathways may allow human cells to survive in embryos of species more suitable for regenerative medicine for health problems.

However, the findings also stated that the human and monkey cells did not mesh perfectly. Human cells often stuck together, leading researchers to wonder if there is another barrier they are unaware of that would prevent human cells from thriving if the embryos mature further.

Chimaeras consisting of humans and monkeys do raise some concerns.

Last week, the National Academies of Sciences, Engineering, and Medicine released a report suggesting that human nerve cells may enter the brains of animals and alter their mental capabilities [7].

In this study, however, there are no nervous systems in the chimaeras. Therefore, it is impossible for them to experience pain and not be conscious. However, experts believe the story would be very different if human-monkey chimaeras were permitted to develop further - suggesting that it could go out of their hands.

"My first question is: Why?" Kirstin Matthews, a science and technology fellow at Rice University's Baker Institute, said, "I think the public is going to be concerned, and I am as well, that we're just kind of pushing forward with science without having a proper conversation about what we should or should not do" [8].

According to the researchers, they do not intend to implant any hybrid embryos into monkeys. Instead, the goal is to better understand how different cells communicate with one another during the early stages of embryonic development.

Several scientists question the need for such experiments using closely related primates - these animals are unlikely to be used as model animals in the same way as mice and rodents. Researchers are concerned that such work may stoke public opposition as non-human primates are protected by stricter research ethics rules than rodents [9][10].

Although there are concerns, scientists hope that these human-animal hybrids, known as chimaeras, can provide better models for testing drugs and growing human organs for transplantation.

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Story first published: Tuesday, August 9, 2022, 16:35 [IST]

Original post:
First-Ever Embryos With Monkey And Human Cells; Scientists Divided Over The Creation - Boldsky

Grafted hPSC-derived GABA-ergic interneurons regulate seizures and specific cognitive function in temporal lobe epilepsy | npj Regenerative Medicine -…

hMGE cell grafting substantially reduced SRS in chronically epileptic rats (CERs)

The effect of grafting hMGE progenitors expressing the Gi-protein-coupled receptor hM4Di into the hippocampus of CERs was evaluated on SRS activity in the fourth month after grafting through continuous video-electroencephalographic (video-EEG) recordings (Fig. 1). The CERs receiving grafts were immunosuppressed with daily cyclosporine A injections (10mg/kg) starting two days before grafting and continuing until the experimental endpoint to avoid transplant rejection. Ungrafted control CERs also received the same regimen of cyclosporine injections to identify any cyclosporine-induced effects on seizures. The total numbers of SRS and stage V-SRS and the total time spent in seizure activity were measured. Compared to ungrafted CERs, grafted CERs displayed substantial reductions in the number of SRS/hour (76% reduction, p<0.0001, unpaired, two-tailed Students t test Fig. 2a), number of stage V-SRS/hour (87% reduction, p<0.0001, unpaired, two-tailed Students t test, Fig. 2b), and the total time spent in seizure activity (76% reduction, p<0.0001, unpaired, two-tailed Students t test, Fig. 2c). Thus, grafting of hPSC-derived hMGE progenitors into the hippocampus in the chronic phase of TLE significantly reduced both frequency and intensity of SRS.

Quantification in the 4th month after grafting via continuous video-EEG recordings revealed that compared to the group of CERs receiving no grafts, the group of CERs receiving hMGE cell grafts displayed greatly decreased frequencies of all SRS (a) and stage V SRS (b). The grafted animals also spent much less time in seizure activity (c). df illustrate electroencephalographic (EEG) traces during the pre-clozapine-N-oxide (CNO), CNO, and post-CNO periods. Values in bar charts are presented as meanS.E.M. ****p<0.0001 (unpaired, two-tailed Students t test).

Continuous video-EEG recordings before (days 15), during (days 68), and 2 days after (days 1114) silencing the graft-derived GABA-ergic interneurons through CNO injections evaluated the influence of graft-derived interneurons in controlling SRS activity in CERs receiving grafts. Examples of EEG traces during the pre-CNO, CNO, and post-CNO periods are illustrated (Fig. 2df). Because the action of CNO is expected to last 23h after each administration and to avoid effects associated with its accumulation due to repeated administration, we administered CNO once every 8h to activate DREADDs. Also, we employed 2 days of washout period to avoid any trace amounts of CNO interfering with the post-CNO results. Silencing of graft-derived neurons substantially escalated SRS activity in CERs compared to the extent of SRS activity before CNO administration (Fig. 3ac). Overall, one-way analysis of variance (ANOVA) with the NewmanKeuls multiple comparison tests revealed that there was a 1.49.7-fold increase in the frequency of all SRS (p<0.01, Fig. 3a), 1.26.4-fold increase in the frequency of stage V-SRS (p<0.05, Fig. 3b), and 1.36.2-fold increase in the total time spent in SRS activity (p<0.05, Fig. 3c). Then, the effect of CNO washout on SRS activity was evaluated two days after the last CNO injection. All parameters of SRS activity were restored to pre-CNO levels. One-way ANOVA with Newman-Keuls multiple comparison tests showed that compared to the CNO period, the frequencies of SRS and stage V-SRS were reduced by 5771% (p<0.01, Fig. 3a, b), and the time spent in seizure activity was reduced by 60% (p<0.05, Fig. 3c).

The bar charts ac compare all SRS and stage V SRS frequencies and times spent in SRS activity (% of recorded time) during pre-CNO, CNO, and post-CNO periods. The bar charts df compare all SRS and stage V SRS frequencies and times spent in SRS activity during the pre-CNO (days 15), CNO (days 68), and post-CNO (days 1114) periods. The bar chart g compares the average electroencephalographic (EEG) power (i.e., spectral density) recorded in interictal periods during pre-CNO, CNO, and post-CNO phases. Values in bar charts are presented as meanS.E.M. *p<0.05; **p<0.01; NS, non-significant (one-way ANOVA with NewmanKeuls multiple comparisons test).

We also evaluated the seizure parameters/day in the pre-CNO (days 15), CNO (days 68), and post-CNO periods (days 1114; Fig. 3df) using one-way ANOVA with the NewmanKeuls multiple comparison tests. In CERs receiving grafts, the total SRS and stage-V SRS/day and the time spent in SRS activity/day were lower in the pre-CNO period. There was no difference in seizure activity over five days in this phase (p>0.05). The administration of CNO enhanced the total SRS and stage-V SRS/day and the time spent in SRS/day. Furthermore, comparable seizure activity was seen over the 3-day CNO period (p>0.05; Fig. 3df). The total number of all SRS on day 7 in the CNO period was higher than all SRS on pre-CNO days 15 (p<0.05; Fig. 3d). Also, the number of stage-V SRS on day 7 in the CNO period was significantly higher than stage-V SRS recorded on pre-CNO days 1 and 3 (p<0.05; Fig. 3e). Notably, all parameters of seizures/day declined in the post-CNO period after two days of CNO washout (Fig. 3df). Also, there was no difference in seizure activity during the four-day post-CNO period (p>0.05). The total numbers of all SRS on days 1214 in the post-CNO period were significantly lower than all SRS recorded on day 7 in the CNO period (p<0.05; Fig. 3d). Additionally, all parameters of seizures were comparable between pre-CNO (days 15) and post-CNO (days 1114) periods (p>0.05; Fig. 3df), implying that the inhibitory function of graft-derived interneurons is restored after the CNO washout period.

Furthermore, we performed spectral analysis of EEG activity in interictal periods by measuring randomly chosen thirty-minute duration interictal segments devoid of noise signals (610 segments/animal, n=5/group). One-way ANOVA with the Newman-Keuls multiple comparison tests revealed that compared to the pre-CNO period, the average EEG power enhanced in the CNO period (p<0.05, Fig. 3g). However, following the CNO washout, the EEG power declined substantially (p<0.05, Fig. 3g). Also, the percentage of waves is significantly reduced in the CNO period compared to the pre-CNO period (meanS.E.M., pre-CNO period 17.53.0; CNO period, 8.71.2; p<0.05) but increased following CNO washout (111.9). Overall, in addition to enhancing the frequency and intensity of SRS, silencing graft-derived GABA-ergic interneurons through CNO injections resulted in enhanced interictal activity, which subsequently waned after the CNO washout.

Next, to examine the direct effect of CNO on SRS activity, we measured SRS activity in CERs that did not receive grafts with CNO administration. One-way ANOVA with the NewmanKeuls multiple comparison tests demonstrated that the frequencies of all SRS and stage V-SRS and the time spent in SRS activity remained comparable across pre-CNO, CNO administration, and post-CNO periods (p>0.05, Fig. 4ac). Thus, in CERs receiving hMGE cell grafts, SRS activity increased when graft-derived GABA-ergic interneuron function was blocked, implying the direct involvement of graft-derived interneurons in seizure control. Furthermore, CNO alone did not affect SRS activity, as CNO administration in CERs receiving no grafts did not change all SRS and stage V-SRS frequencies or the time spent in SRS activity.

The bar charts ac compare all SRS and stage V SRS frequencies and times spent in SRS activity (% of recorded time) during the pre-CNO, CNO, and post-CNO periods in CERs receiving no grafts. Values in bar charts are presented as meanS.E.M. NS, non-significant (one-way ANOVA with NewmanKeuls multiple comparisons test).

We employed an object location test (OLT) to examine the cognitive ability of animals to detect subtle changes in their immediate environment (Fig. 5a), a function linked to normal network activity in the hippocampus36,37. In OLT, the animals with altered hippocampal circuitry/dysfunction consistently show an inability to detect minor alterations in the environment. Naive control rats recognized the change that occurred in the position of one of the objects by exploring the object in the novel place (OINP) for significantly greater periods than the object that remained in the familiar place (OIFP, p<0.0001, unpaired, two-tailed Students t test, Fig. 5b) in trial-3 (T3). In contrast, CERs receiving no grafts showed impaired cognitive function by spending nearly equal amounts of their object exploration time with the OINP and the OIFP (p>0.05, unpaired, two-tailed Students t test, Fig. 5c). Notably, CERs receiving hMGE cell grafts behaved similarly to naive control rats by showing a greater affinity for the OINP than the OIFP (p<0.01, unpaired, two-tailed Students t test, Fig. 5d). These results suggest that grafting of hPSC-derived hMGE cells into the hippocampus could alleviate chronic epilepsy-related object location memory impairment.

a depicts the various trials involved in an object location test (OLT). The bar charts in be compare percentages of time spent with the object in the familiar place (OIFP) and the object in the novel place (OINP) in naive control rats (b), chronically epileptic rats (CERs; c), and CERs with hMGE grafts before and during the clozapine-N-oxide (CNO) treatment (d, e). The bar chart in f compares the time spent with the OINP across the four groups with ANOVA. Object location memory was impaired in CERs with no grafts and CERs with grafts when graft-derived interneurons were silenced. g shows the various trials involved in a pattern separation test (PST). The bar charts in hk compare percentages of time spent with the familiar object on pattern 2 (FO on P2) and the novel object on pattern 2 (NO on P2) in naive control rats (h), CERs (i), and CERs with hMGE grafts before and during the clozapine-N-oxide CNO treatment (j, k). The bar chart in l compares the time spent with the NO on P2 across the four groups with ANOVA. Note that pattern separation ability was impaired in CERs with no grafts. However, CERs with grafts displayed pattern separation ability even when the graft-derived interneurons were silenced with CNO. Values in bar charts are presented as meanS.E.M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS, non-significant (be, hk, unpaired, two-tailed Students t test; f, l, one-way ANOVA with NewmanKeuls multiple comparisons test).

To investigate the role of graft-derived GABA-ergic interneurons in the object location memory task, we silenced the transplant-derived DREADDs expressing interneurons through CNO administration and performed the OLT with new objects. With the silencing of transplant-derived interneurons, CERs lost their ability to distinguish the OINP from the OIFP, which was evident from their exploration of OINP and OIFP for almost equal periods (p>0.05, unpaired, two-tailed Students t test, Fig. 5e). The parameters such as total object exploration times, distances traveled, or movement velocities were comparable between the pre-CNO and CNO periods (data not illustrated). Comparison of the time spent with the OINP across groups (naive, CERs, CERs + grafts in the pre-CNO and CNO periods) using one-way ANOVA with the Newman-Keuls multiple comparison tests revealed impaired object location memory in CERs with no grafts, and CERs with grafts when graft-derived interneurons were silenced (Fig. 5f). However, in the absence of CNO, CERs with grafts displayed similar object location memory as naive control rats. Thus, graft-derived GABA-ergic interneurons in CERs directly influenced the object location memory function, a hippocampus-dependent cognitive task.

The pattern separation test (PST) examines proficiency in discriminating similar experiences by storing similar representations in a non-overlapping manner and is linked to the dentate gyrus and adult hippocampal neurogenesis38,39. For this test, the movement of each rat was investigated in an open field with four successive trials (5min each), separated by 30-min intervals. The first three trials successively involved the exploration of an open field apparatus (T1), a type of identical objects placed on a floor pattern 1 (P1; T2), and the second type of identical objects placed on a floor pattern 2 (P2; T3). In T4, the animal explored objects on P2, with one of the objects from T3 replaced with an object from T2. The object from T2 is a novel object on pattern 2 (NO on P2), whereas the object retained from T3 is a familiar object on P2 (FO on P2) (Fig. 5g). Naive control animals displayed a greater propensity to explore the NO on P2 than the FO on P2 in T4 (p<0.001, unpaired, two-tailed Students t test, Fig. 5h). CERs receiving no grafts displayed a pattern separation deficit, which was evident from their lack of interest in exploring the NO on P2 for higher periods than the FO on P2 (p>0.05, unpaired, two-tailed Students t test, Fig. 5i). In contrast, CERs receiving grafts showed similar behavior as naive control animals by displaying a greater propensity to explore the NO on P2 than the FO on P2 (p<0.01, unpaired, two-tailed Students t test, Fig. 5j). Thus, grafting of hPSC-derived hMGE cells into the hippocampus alleviated chronic epilepsy-induced pattern separation dysfunction.

To determine whether graft-derived interneurons played a role in restoring the pattern separation function in CERs, we silenced the transplant-derived DREADDs expressing interneurons through CNO administration and performed the PST with new objects and floor patterns. With the silencing of transplant-derived interneurons, CERs did not lose their ability to distinguish the NO on P2 from the FO on P2, which was evident from their exploration of the NO on P2 for higher periods than FO on P2 (p<0.01, unpaired, two-tailed Students t test, Fig. 5k). Furthermore, the parameters such as total object exploration times, distances traveled, or movement velocities were comparable between the pre-CNO and CNO periods (data not illustrated). Comparison of the time spent with the NO on P2 across groups (naive, CERs, CERs + grafts in the pre-CNO and CNO periods) using one-way ANOVA with the Newman-Keuls multiple comparison tests revealed impaired pattern separation function in CERs with no grafts, but not in CERs with grafts even when graft-derived interneurons were silenced (Fig. 5l). Thus, CERs with grafts displayed similar pattern separation ability as naive control rats in the absence and presence of CNO, implying that graft-derived GABA-ergic interneurons in CERs did not directly influence the pattern separation function.

Stereological quantification of HNA+ cells per hippocampus revealed that the overall graft cell yield is >800,000 cells/hippocampus (meanS.E.M=886,26655,967, n=4). Since the graft cell yield per hippocampus was higher than the number of cells initially injected (~300,000 live cells in 3 grafts, ~100,000 cells/graft), the results implied that the grafted progenitors divided a few times after grafting as some donor cells likely die during transplantation.

To confirm DREADD expression in transplant-derived cells, we performed immunofluorescence studies on tissue sections through the hippocampus to visualize human nuclear antigen (HNA, a marker of grafted human cells) and neuron-specific nuclear protein (NeuN, a marker of neurons). Confocal microscopic analyses of HNA and mCherry (the reporter of DREADD expression) revealed that virtually all HNA+ cells in grafts expressed DREADDs (Fig. 6ac). Similar analysis of NeuN and mCherry showed that all neurons within grafts expressed DREADDs (Fig. 6df). The hESC line employed in the study was built by inserting a construct of DREADD and mCherry separated by 2A. Furthermore, the expression of DREADD and mCherry in the cell line is under the control of the universal CAG promoter, and hence mCherry is expressed stably in all cells. Earlier grafting studies have demonstrated similar results using this cell line35,40.

Note that mCherry is displayed in virtually all HNA+ graft-derived cells (ac), NeuN+ neurons (df), and GABA-ergic interneurons (jl). gi demonstrate that a vast majority (meanS.E.M, 80.81.1%) of HNA+ graft-derived cells differentiated into GABA-ergic interneurons. Scale bars: al, 20m.

Next, we determined the differentiation of graft-derived cells into NeuN+ neurons or GABA+ interneurons through HNA and NeuN, or HNA and GABA dual immunofluorescence and Z-section analysis in a confocal microscope. Such quantification demonstrated that ~85% of HNA+ expressed NeuN (meanS.E.M=85.21.2, n=6) and ~81% of HNA+ cells expressed GABA (meanS.E.M=80.81.1%, n=6). Examples of hMGE cells differentiating into GABA-ergic interneurons are illustrated (Fig. 6gi). The overall differentiation is consistent with our earlier grafting study using hiPSC-derived MGE cells as donor cells in an SE model22. Next, to confirm the expression of DREADDs in graft-derived GABA-ergic interneurons, we examined mCherry expression in these interneurons. Virtually all GABA-ergic interneurons expressed mCherry (Fig. 6jl). In addition, transplanted hMGE cells also differentiated into subclasses of GABA-ergic interneurons expressing PV or NPY, which also displayed DREADDs (Fig. 7af). These results suggest that CNO administration could block the function of graft-derived interneurons because of their robust expression of DREADDs.

Gi-protein-coupled receptor hM4Di expression (with mCherry reporter) in parvalbumin (PV) and neuropeptide Y (NPY) expressing interneurons derived from human medial ganglionic eminence progenitor cell grafts in the hippocampus of chronically epileptic rats (af), and putative synapse formation between graft-derived axons and host neurons (gp). Note that mCherry is apparent in PV and NPY+ interneurons derived from graft-derived cells (af). g, l illustrate putative synapse formation between graft-derived presynaptic boutons (green colored structures expressing human synaptophysin (hSyn) and the host postsynaptic density protein 95 (PSD95, red particles) elements on microtubule-associated protein-2 (MAP-2) positive dendrites (blue) in the host CA1 stratum radiatum (g) and the dentate gyrus molecular layer (l). h, m are magnified views of boxed regions in g, l, respectively. ik, np illustrate MAP-2, hSyn, and PSD95 elements in red, green, and blue channels. Scale bars: af, 20m; g, l, 5m; ik, np, 0.5m.

Enhanced frequency and intensity of SRS following silencing of graft-derived GABA-ergic interneurons expressing DREADDs implied connectivity between hMGE graft-derived GABA-ergic interneurons and the host neurons. To confirm this, we employed Z-section analyses in a confocal microscope of brain tissue sections through the hippocampus processed for triple immunofluorescence to localize the human-specific synaptophysin (hSyn, the presynaptic protein in graft-derived neurons), postsynaptic density protein-95 (PSD-95), and microtubule-associated protein-2 (MAP-2) in soma and dendrites of host neurons. Such analysis suggested the formation of putative synaptic contacts by graft-derived neurons on the dendrites of host CA1 pyramidal neurons in the stratum radiatum (Fig. 7g) and dentate granule cells in the molecular layer (Fig. 7l). Magnified views showing the possible contacts between the presynaptic component derived from graft-derived interneurons (h-Syn+ structures in green) and the host postsynaptic component (PSD95+ structures in blue) on the dendrites of CA1 pyramidal neurons and dentate granule cells (in red) are illustrated (Fig. 7hk, mp). In addition, hSyn+ structures were also seen on the soma of dentate granule cells. Thus, transplanted GABA-ergic interneurons appeared to have integrated synaptically with the host neurons in the dentate gyrus and the CA1 subfield. Such synaptic connectivity likely explains the control of seizures and object location memory task by transplant-derived GABA-ergic interneurons.

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Grafted hPSC-derived GABA-ergic interneurons regulate seizures and specific cognitive function in temporal lobe epilepsy | npj Regenerative Medicine -...