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Purification technologies for induced pluripotent stem cell therapies - Nature.com

Finishing the odyssey to a stem cell cure for type 1 diabetes – Nature.com

A recent clinical study by Pipeleers and colleagues has brought the possibility of a stem-cell based cure one step closer1. This perspective will summarize the major hurdles that have been overcome to deliver cell-based improvements in glucose control and highlight the key issues that stand between this important proof-of-concept clinical study and a durable cure for the majority of patients living with T1D.

The autoimmune destruction of pancreatic cells creates a lifelong dependence on insulin to control blood sugar levels in individuals with type 1 diabetes (T1D). Over time, poorly managed T1D causes microvascular and macrovascular complications that significantly impact quality of life2. Unfortunately, intensive glucose lowering therapy to reduce these long-term complications of hyperglycemia is accompanied by an increased risk of hypoglycemic events3. Technological solutions aiming to replace cell function with an artificial pancreas can improve glucose control by integrating continuous glucose monitoring with automated insulin delivery4. However, these systems have not yet matched the exquisite blood glucose control provided by human islets5, and T1D patients remain burdened with the ongoing management and expense of a chronic disease.

Therapeutic approaches aimed at restoring a functional -cell mass could eventually eliminate the need for exogenous insulin. Indeed, transplant of cadaveric islets into immunosuppressed T1D recipients has shown that excellent glucose control can be achieved6, while simultaneously reducing hypoglycemic risk7. The benefits of islet transplant to individual T1D islet recipients should not be minimized, however, the limited supply of donor tissue constrains the potential impact of this strategy, which is still only available to clinical trial participants in many countries including the USA. In contrast, human pluripotent stem cells (hPSCs)8,9 could theoretically be expanded and differentiated to restore a functional -cell mass in all eligible patients with T1D if they can be shielded from autoimmune attack.

Initially, a major goal was to optimize stem cell differentiation protocols to produce glucose-responsive cells from hPSCs. The first major success was guided by developmental studies from diverse model organisms10, in which step-wise modulation of key developmental signals produced cells capable of expressing insulin11, albeit at low levels and in a largely constitutive manner. Nevertheless, this was a remarkable demonstration that hPSCs have the potential to be used for cell-replacement therapy. Extensive empirical optimization and an appreciation of the functional importance of islet structure led to -cells with improved function12,13. We note, the in vitro generation and characterization of stem-cell derived islets has been recently reviewed14. However, the observation that in vitro differentiated hPSC-derived -cells exhibit immature physiological responses15, like many other hPSC-derived cell products16, led to consideration of alternative strategies. A surprisingly effective approach has involved halting in vitro differentiation once pancreatic fate is established at the multipotent pancreatic progenitor (PP) stage and allowing -cell differentiation and functional maturation to be guided by endogenous cues post-transplant17. An added benefit of this approach is that PP differentiation is amenable to the large-scale expansion and Good Manufacturing Practice (GMP) production and quality control required for clinical application18. Interestingly, further differentiation and enrichment of hormone-positive islet-like cells prior to transplant does not reduce the in vivo maturation time19.

Now that a suitable cell-source is available, preventing graft rejection is one of the greatest challenges facing hPSC-based therapies. The autoimmune nature of T1D poses a challenge for cell-based therapies since the immune system is poised to destroy newly transplanted material, even if it is derived from the patients own stem cells. As seen with cadaveric transplants, systemic immunosuppression can protect and maintain unmatched donor cells in a functional state6. Furthermore, clinical transplants have shown that ~10,000 islet equivalents/kg provide a functional -cell mass that can eliminate the need for exogenous insulin20, setting a clear goal for therapeutic effect. Unfortunately, this blunt force approach trades dependence on insulin for continuous immunosuppression, which brings increased risks of infections, certain cancers and regimen-specific toxicities21.

Encapsulating transplants in biocompatible materials that prevent immune infiltration, while permitting sufficient diffusion of nutrients and waste products to support -cell health, has been pursued to eliminate the need for systemic immunosuppression. Despite the demonstration over 40 years ago that microencapsulation is sufficient to preserve islet function for several weeks in an animal model without immune suppression22, maintaining a functional -cell mass within cell-impermeable materials remains a major challenge. Microencapsulated islets (single islets or small clusters) can disperse into the recipient tissue where they benefit from a large contact area with the host. However, the impermeable barrier prevents direct contact with blood vessels, which produce a basement membrane that is likely essential for optimal -cell function23. These problems have been even more pronounced in cell-impermeable macroencapsulation devices, where elaborate designs such as intravascular hollow fibers are used to increase exposure to the bloodstream24. However, despite the theoretical advantages of close contact with the blood stream, the serious risk of blood clots associated with vascular prostheses has impeded clinical translation of intravascular devices25. The strengths and weaknesses of additional islet encapsulation technologies have been recently reviewed26.

Because cell impermeable materials necessarily prevent direct contact between -cells and the endothelium, some groups have gone a different direction with cell-permeable devices, including Viacyte with the VC-02 device. Although the exact configuration of the VC-02 remains proprietary, key features that appear to have contributed to clinical success are a perforated encapsulation membrane that is encased in another layer of perforated non-woven fabric27. The VC-02 device loaded with hPSC-derived Pancreatic Endoderm Cells (PECs) that are partially differentiated to the PP stage has been coined the PEC-Direct (Fig. 1).

Partially differentiated hPSC-derived PECs were loaded into devices that mature under the protection of systemic immunosuppression in T1D patients. The perforated design facilitates the infiltration of endothelial cells, while the external non-woven fabric restricts fibrotic foreign body responses. After maturation, functional cells comprised 3% of the total cell mass. MO macrophage, T T cell, NK Natural Killer cell.

While most clinical experience is associated with transplant of cadaveric islets to the portal vein in the liver, additional subcutaneous, omental, and intramuscular sites have been extensively studied in preclinical models28. These sites may pose additional challenges for islet survival since the limited clinical data available suggests that unencapsulated extrahepatic transplants do not perform well29. However, encapsulated hPSC-derived PPs transplanted subcutaneously differentiate into tissue that contains functional glucose responsive cells within 4-6 months in animal models30,31. Building on this experience, two parallel first-in-human studies aimed to optimize the cell dose and perforation configuration of PEC-Direct subcutaneous implants in small numbers of T1D recipients (n=1732; n=1533) demonstrated that C-peptide, a marker produced by insulin-secreting cells, could be newly detected in some individuals at 6 months post-transplant and could persist until 24 months. A subset of patients achieved >30 pM C-peptide after meal stimulation (6/24, note some individuals were analyzed in both studies), a level that is associated with reduced T1D complications34. However, none of the individuals reached the 200 pM threshold associated with improved metabolic control34 or the 1000 pM level associated with insulin independence in cadaveric islet recipients35. For reference, postprandial C-peptide levels range from 1000-3000 pM in healthy individuals36. Importantly, the observed insulin production could be directly attributed to the VC-02 devices, and not the recovery of the recipients own cell function, since removal of the explants eliminated the improvements in C-peptide levels in two patients where this was carefully explored33. While comparison of transplanted PEC cells with cadaveric islets in terms of islet equivalents can only be approximated, these pilots delivered at most one-half the transplant volume required for insulin independence. Since the recovered devices contained mostly glucagon+ cells (16%) and only a small fraction of insulin+ cells (3%) it is not surprising that the transplants were not sufficient to improve secondary measures of glycemia. Regardless, these first-in-human studies demonstrated the overall safety of the approach in high risk (hypoglycemia unaware) patients with all serious adverse events attributed to the immunosuppressive regimen or surgical procedure, suggesting that maximizing transplant size and -cell composition were going to be crucial for clinical impact.

In an interim report of 1-year outcomes, Keymeulen et al., now provide evidence that hPSC transplants are on the cusp of providing benefit to many patients. Using an adaptive trial design, the transplant volume was increased 2-3 fold and all devices used the perforation pattern and density associated with the best outcomes in previous trials32,33 The transplant recipients were selected using similar criteria to the previous trials, requiring stable T1D (>5 years), a high risk for hypoglycemic complications (Clarke score 4), and meal-stimulated C-peptide levels 30 pM prior to transplant. With the increased dose and optimized device configuration, 3/10 recipients produced 100 pM postprandial C-peptide from 6-months post-transplant and one surpassed the 200 pM threshold associated with metabolic significance. Excitingly, this individual achieved improved time spent in the target blood sugar range (by continuous glucose monitoring), a clinically meaningful measure of function.

Now that hPSC-derived cells have been shown to produce metabolically significant amounts of insulin in a T1D patient, there is a path to match and potentially exceed the outcomes observed with cadaveric transplants. Assuming a linear relationship between -cell mass and insulin secretion, it appears that a further ~10-fold increase in functional -cell mass would be sufficient to achieve insulin-independence (>1000 pM C-peptide) in some patients and a metabolic benefit (>200 pM C-peptide) in most recipients. Unfortunately, simply further increasing the transplant size would likely increase surgical complications. Consistent across the clinical trials, recovered PEC-Direct devices contained large acellular regions filled with extracellular matrix. This material permanently occupies space that could be better utilized as cells currently comprise at most ~3% of the total volume within a device1. Although histological analysis of the PEC-Direct devices retrieved from the non-responders was not available in the interim report, further insight into the fate of transplanted PPs and the composition of infiltrating cells in failed grafts will help focus future efforts. Interestingly, in samples from two responders, the less functional graft was already dominated by infiltrating recipient cells at 3 months post-transplant and the cell mass was negligible at 9 months despite having a larger total cell volume1. Human islets are composed of ~50% cells that are interspersed with other endocrine cell types and aligned to the vasculature37. Thus, if the majority of the device volume were filled with islet-like structures, there should be a sufficient functional cell mass for most patients.

Recapitulating embryonic pancreatic development in vitro has produced PPs that clearly have the potential to complete differentiation into functional cells in a process that takes 4-6 months post-transplant. Additional clues from developmental biology indicate that there are stage-specific interactions between endogenous endocrine precursors and the vasculature that influence pancreatic differentiation. Initially, endothelial cells induce the differentiation of endocrine cells38, which then signal back to increase the density of the local vascular network39 and deposition of a vascular basement membrane that promotes cell function23. Thus, cells participate in the construction of a specialized niche through interactions with the vasculature that are essential for subsequent cell maturation. While the perforated design of the PEC-Direct device allows infiltration of endothelial cells, the growth of this vascular network takes time and is competing with recipient fibroblasts which are only partially blocked by the outer non-woven fabric layer (Fig. 1), suggesting that there are limitations to mechanical control of these processes. The strengths and weaknesses of the PEC-Direct device compared to other cell-based therapies are summarized in Table 1. Here, we highlight recent advances that could help maximize the yield of vascularized cells and in the best-case scenario provide an immune privileged niche that would eliminate the need for systemic immunosuppression (Fig. 2).

Immunomodulatory materials and cells could be used to create an immune privileged niche for transplanted PECs and further discourage fibroblast infiltration. cell numbers could potentially be increased by improving the microenvironment and converting other pancreatic cell types to the cell fate. MO macrophage, T T cell, NK Natural Killer cell. Treg Regulatory T cell, M2 M2 macrophage, CXCL12 CXCL12 chemokine.

The materials in the PEC-Direct device, particularly the outermost non-woven fabric layer, suppress a full-blown foreign body response associated with the recruitment of macrophages and fibroblasts to the interface with recipient tissues27. Limiting residual fibroblast infiltration1 might be most important in the acute post-transplant period, as they likely interfere with PP differentiation and the establishment of the intra-device vascular network. The precise composition of the perforated VC-02 encapsulation membrane remains proprietary. However, if it is composed of alginate or similar material, then biomodulatory factors could be directly integrated into the encapsulation membrane40. Notably, incorporation of the CXCL12 chemokine was recently shown to protect microencapsulated xenogeneic islets in a non-human primate model41. The primary mechanism of acute islet protection is associated with repulsion of islet-reactive effector T cells42. However, CXCL12 has multiple immune modulatory roles43, and protected islets also show reduced macrophage and fibroblast surface infiltration and collagen deposition40,41. These studies suggest that incorporating chemokine(s) such as CXCL12 into the encapsulation membrane, or potentially adding an additional biomodulatory layer, could improve the microenvironment within the device. Additional advances in biomaterials functionalized with diverse immunomodulatory molecules have been recently reviewed in the context of islet transplantation44.

Giving the vasculature a head start could be a complementary way to limit the opportunities for intra-device fibrosis. Instead of relying exclusively on the recipients vasculature, the addition of ready-made microvessels isolated from adipose tissue to hPSC-derived PPs improved early graft survival and reduced the time required for cell differentiation to less than 10 weeks in mouse T1D models45. Harvesting recipient microvessels would add additional complexity to a clinical transplant program but a proof-concept pilot study using healthy donor microvessels could be informative. Ideally, microvessel-equivalents would also be produced from hPSCs46, although scale up under GMP conditions as was done for PPs18 would also be needed.

Improving the intradevice microenvironment might increase not only the mature pancreatic cell volume within a device but potentially also the proportion of cells. In the small number of recovered grafts that have been analyzed histologically, cells comprise at most ~3% of the total cell volume1,33,34. In contrast, preclinical studies with similar device-encapsulated PPs have produced grafts with up to 16% cells by transplanting into a preformed pouch at the surgical site47. Presumably, the 5 weeks between pouch formation and device engraftment allowed for vascularization of the transplant site and resolution of acute inflammatory responses. Importantly, these data indicate that partially differentiated PPs are capable of producing significantly more cells within an optimized microenvironment. Beyond improving the host environment, an attractive source of additional cells is from transdifferentiated cells, which are invariably the most abundant pancreatic cell type identified after in vivo maturation of PPs1,47. While paracrine signals from cells are important for optimal cell function48, these intraislet interactions are unlikely to be compromised by the transdifferentiation of excess cells that are currently produced in superphysiological proportions. Furthermore, reducing cell content in the graft could have metabolic benefits as there is growing evidence that hyperglucagonemia interferes with cell function49. cells have an innate ability to transdifferentiate, although it is only triggered by near complete cell destruction50,51. Overexpression of the key cell transcription factors PDX1 and MAFA in adult cells produces -like cells with the ability to sense glucose and secrete insulin52,53, although these cells retain aspects of their previous cell identity. To avoid perturbing differentiation to the PP stage in vitro, implementing directed transdifferentiation in hPSC-derived transplants will require engineered stem cells with the ability to induce cell factors specifically in mature endocrine cells54. A further 2-3 fold increase of the cell mass observed in preclinical studies via transdifferentiation would produce structures with very similar cellular composition to endogenous human islets37.

The ultimate goal of a hPSC-based therapy for T1D is to provide long-term cell function without the need for systemic immunosuppression. Cotransplantation of microgels containing individual immunomodulatory factors such as PD-L155 or FasL56 can have profound effects on graft survival in immunocompetent hosts. For example, FasL presenting microbeads combined with only two weeks of rapamycin monotherapy supported graft function for over six months and induced Treg-dependent local tolerance without systemic effects on the immune system in an allogeneic mouse model56. Similar effects were seen in non-human primates57, although the long-term viability of the grafts was not evaluated.

In addition to achieving allogeneic graft tolerance, hPSC-derived cells must contend with the dysregulated autoimmune response in T1D. Excitingly, it now appears possible to fully cloak hPSCs and their differentiated progeny by overexpressing a cocktail of 8 immunomodulatory factors that includes PD-L1 and FASL58. Together, these factors disrupt antigen presentation, T-cell and NK cell attack, and innate inflammatory responses. By activating a proliferation-dependent kill switch59, cloaked cells could be maintained in a dormant state within an immunocompetent host. Furthermore, these cloaked cells protected their neighbors, including allogeneic islets and xenogeneic hPSCs. While PPs could potentially be generated directly from cloaked hPSCs, the overexpression of 8 genes might impact cell function long-term. An elegant strategy to address all the key issues discussed here would be to generate cloaked endothelial cells for cotransplantation with PPs that are genetically primed for to cell transdifferentiation.

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Finishing the odyssey to a stem cell cure for type 1 diabetes - Nature.com

‘We can’t answer these questions’: Neuroscientist Kenneth Kosik on whether lab-grown brains will achieve consciousness – Livescience.com

Brain organoids are 3D, lab-grown models designed to mimic the human brain. Scientists normally grow them from stem cells, coaxing them into forming a brain-like structure. In the past decade, they have become increasingly sophisticated and can now replicate multiple types of brain cells, which can communicate with one another.

This has led some scientists to question whether brain organoids could ever achieve consciousness. Kenneth Kosik, a neuroscientist at the University of California, Santa Barbara, recently explored that possibility in a perspective article. Live Science spoke with Kosik about how brain organoids are made, how similar they are to human brains and why he believes that brain organoid consciousness is not likely anytime soon.

Related: In a 1st, 'minibrains' grown from fetal brain tissue

EC: What are brain organoids, and how do scientists make them?

Kenneth Kosik: A brain organoid is made from stem cells. You can take any person and convert their, say, skin fibroblasts into stem cells, and then differentiate them into neurons. It's what stem cells are all about stem cells are called "pluripotent" because they can make any cell in the body.

We spent a fair amount of time before organoid technology came along, taking human-induced pluripotent stem cells and inducing them in a two-dimensional array to look at neuronal differentiation.

So that takes us halfway there. But it only gets us as far as two dimensions. And then the big insight, which came from Yoshiki Sasai in Japan and Madeline Lancaster, was to take these neurons that were beginning to differentiate cells relatively early in development and put them in a drop of what's called Matrigel a gel that can be either a liquid or a solid depending on the temperature.

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So the cells are in this drop, and then the magic happens. Instead of growing in two dimensions, they start to grow in three dimensions. It absolutely fascinates me that when biology begins to explore the third dimension, a very novel biology emerges. Certainly, in two dimensions, these neurons that were growing could achieve a very wide diversity of cell types, but they did not achieve any kind of interesting anatomy.

Once they're growing in three dimensions, they start to form relationships to each other, kinds of structure and anatomy, that has a very loose resemblance to the brain. And I really emphasize the word "loose," because there are people that use a misnomer for brain organoids and call them "minibrains." They're not brains at all. They are organoids meaning like the brain.

A question we're keenly interested in, and many labs are, is that if organoids are like the brain, to what degree do they resemble the brain and to what degree do they differ? And they differ from the brain a lot, so you have to be very careful about interpretations of organoids. Not everybody thinks that organoids are going to be informative for neuroscience because what we find in an organoid may be over-interpretation. But on the other hand, [it] is forming a three-dimensional structure that has some degree of lamination [formation of layers of cells within tissue], it has these rosettes in which, from the center of the rosette, you can progressively see cells becoming more mature as development proceeds, which is very similar to what happens in the brain.

Related: Lab-grown 'minibrains' may have just confirmed a leading theory about autism

EC: Are there any brain organoids that accurately capture the whole brain yet?

KK: There is no organoid that captures the whole brain. There are approaches that attempt to capture more of the brain than, say, just the one part that maybe we and other labs are working on. These are called "assembloids." [Scientists] take stem cells and differentiate them down a pathway that may make a little more ventral [front part of the] brain, or a little more dorsal [back part of the] brain, and they put them together, they fuse them, so that you get more comprehensive fusion a wider representation, I should say, of brain anatomy.

There are other ways of making organoids that are a little more indiscriminate. They're not directing the stem cells towards dorsal and ventral, they are putting them all together. That's a lot of what we do. Those were the techniques that were originated by Lancaster. And in that case, it's my opinion that when you do it that way, you get a broader representation of cell types. That's what you gain, but you sacrifice anatomical accuracy because when you make an assembloid, the anatomy is not great. But when you do it without differentiating toward dorsal and ventral and you put it all together, the anatomy becomes even more problematic.

EC: As you alluded to, these organoids are similar to human brains, but there's some key physiological differences. Can you explain those?

KK: So, one similarity right away is that you see a lot of spiking going on.

(Editor's note: Kosik is referring to the fact that, when an organoid is hooked up to electrodes, this triggers electrical spikes, or signals, transmitted between neurons.)

It's quite remarkable, and underlying this is the notion, which is probably what intrigues me the most, that all of this activity is spontaneous: it just arises based on the assembly of the neurons.

And now we can look at the relationships of those spikes. When you do that, you can ask the question, well, if I see neuron A firing, what's the likelihood that I'll see neuron B firing? I'm going to look at the binary relationships among all of them and I'm doing it with the filter that when neuron A fires I'm only going to look at when another neuron fires within 5 milliseconds. Why 5 milliseconds? Because that's about the time in which it takes for transmission to occur across the synapse. (Editor's note: A synapse is the gap between two neurons.)

And when we do that, you can see that they form a network. You connect A and B, and then you connect C and D, and then A and C. You can see that the neurons are talking to each other and this arises spontaneously.

That is one example of the way in which an organoid does something that will spontaneously resemble what happens in the brain.

The way I look at an organoid, it is a vehicle that has the capacity to encode experience and information if that experience were available to it but it's not. It has no eyes, ears, nose or mouth nothing's coming in. But the insight here is that the organoid can set up spontaneous organization of its neurons so that it has the capacity to encode information, when and if it becomes available. That's just a hypothesis.

Related: Lab-grown 'minibrains' help reveal why traumatic brain injury raises dementia risk

EC: Do you think that brain organoids will ever achieve consciousness?

KK: So that's where things get a little mysterious. I think that those kinds of questions are predicated on this term that people have a lot of trouble defining: consciousness.

[Based on currently fashionable theories of consciousness] I would say, "No, it doesn't even come close."

Related: Mini model of human embryonic brain and spinal cord grown in lab

EC: You spoke about the fact that organoids have shown some capacity to encode information, but they don't have the experience to do this in the first place. What would happen if, hypothetically, a human brain organoid was transplanted into an animal? Could it then achieve consciousness?

KK: Let's break that down. Before it is transplanted into an animal, some people would say the animal already has consciousness and some people would say [it does] not. So, right away, we get into this difficulty about where in the animal kingdom does consciousness begin? So, let's reframe the question. If you then took an animal, which may or may not have some degree of consciousness, and you transplant in a human organoid, would you confer consciousness on that animal or would you enhance consciousness, or would you even get something that resembled human consciousness in the animal? I don't know the answer to any of those questions.

We can do these hybrids now so it's a good question. But the evaluation of consciousness now, because of all the problems as to what consciousness is, is still going to be an open question.

Related: Could mini space-grown organs be our 'cancer moonshot'?

EC: Do we have an idea of rough timescales , is consciousness something that could happen in the near future, after, say, a certain number of years, or is it still really uncertain at this point?

KK: Technology is moving very fast. One place where we may begin to push the boundary is in the so-called cyborgs, or organoid interfaces. That would be one direction that could be interesting. Maybe a little bit toward consciousness, but even more so toward developing the implementation of human abilities in one of these synthetic systems.

EC: Can you think of any obvious benefits and drawbacks of these organoids being able to achieve consciousness?

KK: We know so little about neuropsychiatric conditions. Neuropsychiatric drugs are developed without understanding any deep physiology. All of that could be done, I think, with organoids. I think as disease models, it could be very, very useful [for them to achieve consciousness].

The dream state that I have is to develop them as computational systems because, right now, to do the kinds of very expensive computations that are required for ChatGPT and many of these large language models, these take hundreds of millions of dollars to develop. They require a server farm of energy to keep them going. We're really just running out of computer power. Yet, the brain does a lot of this stuff on 20 watts. So, a big interest for me is, "Can organoids, if not solve, contribute to the huge demands that we're making on the energy system by tapping into the highly efficient way in which the brain, and presumably the organoid, can handle information?"

Editor's note: This interview has been edited and condensed.

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'We can't answer these questions': Neuroscientist Kenneth Kosik on whether lab-grown brains will achieve consciousness - Livescience.com

Astellas and Graduate School of Medicine / Faculty of Medicine, Osaka University Enter into Research Collaboration to Develop Pluripotent Stem…

- Collaborative Research on Innovative Cartilage Organoid Cell Therapy for Intervertebral Disc Degenerative Disease using Astellas' Universal Donor Cell Technology -

TOKYOand OSAKA, Japan, July 22, 2024 /PRNewswire/ -- Astellas Pharma Inc. (TSE: 4503,President and CEO: Naoki Okamura,"Astellas") and Graduate School of Medicine / Faculty of Medicine, Osaka University (President: Shojiro Nishio"Osaka University") today announced that Astellas Institute for Regenerative Medicine (a wholly owned subsidiary of Astellas, "AIRM"), Universal Cells (a wholly owned subsidiary of Astellas) and Osaka University have entered into a research collaboration to develop innovative pluripotent stemcell*1-derived cartilage organoid cell therapy for the treatment of intervertebral disc degenerative disease*2.

Universal Cells holds the rights to Universal Donor Cell (UDC) technology to create cell therapy products from pluripotent stem cells that have reduced risk of immune rejection by genetically modifying Human Leukocyte Antigen (HLA) using gene editing technology.

Under the terms of the agreement, the three parties aim to combine the cartilage tissue creation protocol established by Professor Noriyuki Tsumaki of (Graduate School of Frontier Biosciences / Premium Research Institute for Human Metaverse Medicine) the Department of Tissue Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University, a leading researcher in cartilage diseases, Universal Cells' UDC technology, and AIRM's exceptional R&D expertise in cell therapy, and jointly create an innovative cell therapy for intervertebral disc degenerative disease.

Yoshitsugu Shitaka, Ph.D., Chief Scientific Officer (CScO) of Astellas"Astellas is committed to achieving our VISION of being "on the forefront of healthcarechange, turning innovative science into VALUE for patients". We hope to provide our cutting-edge UDC technology to academia and startups globally, and deliver next-generation cell therapies to patients. This partnership is an important step in the open innovation using UDC technology."

Professor Noriyuki Tsumaki, M.D., Ph.D., (Graduate School of Frontier Biosciences / Premium Research Institute for Human Metaverse Medicine) Department of Tissue Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University"We believe that our cartilage-like tissue has the potential to regenerate intervertebral discs. We hope that combining our research with Astellas' UDC technology and R&D cell therapy system will accelerate and realize the development of regenerative therapies to treat intervertebral disc degenerative disease."

*1 Pluripotent stem cell: Cells that possess the ability to proliferate almost indefinitely and differentiate into any cell that makes up the organism. Ex. embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). *2 Intervertebral disc degenerative disease: A type of degenerative spinal disease. The intervertebral discs, which are cartilaginous tissues, play a crucial role as cushions between each bone of the spine by containing a significant amount of water. This helps maintain flexible movement of the back. However, when these discs degenerate, they lose water, resulting in the failure of their cushioning function, which can lead to lower back pain.

About AstellasAstellas Pharma Inc. is a pharmaceutical company conducting business in more than 70 countries around the world. We are promoting the Focus Area Approach that is designed to identify opportunities for the continuous creation of new drugs to address diseases with high unmet medical needs by focusing on Biology and Modality. Furthermore, we are also looking beyond our foundational Rx focus to create Rx+ healthcare solutions that combine our expertise and knowledge with cutting-edge technology in different fields of external partners. Through these efforts, Astellas stands on the forefront of healthcare change to turn innovative science into VALUE for patients. For more information, please visit our website at https://www.astellas.com/en.

Cautionary Notes(Astellas)In this press release, statements made with respect to current plans, estimates, strategies and beliefs and other statements that are not historical facts are forward-looking statements about the future performance of Astellas. These statements are based on management's current assumptions and beliefs in light of the information currently available to it and involve known and unknown risks and uncertainties. A number of factors could cause actual results to differ materially from those discussed in the forward-looking statements. Such factors include, but are not limited to: (i) changes in general economic conditions and in laws and regulations, relating to pharmaceutical markets, (ii) currency exchange rate fluctuations, (iii) delays in new product launches, (iv) the inability of Astellas to market existing and new products effectively, (v) the inability of Astellas to continue to effectively research and develop products accepted by customers in highly competitive markets, and (vi) infringements of Astellas' intellectual property rights by third parties. Information about pharmaceutical products (including products currently in development) which is included in this press release is not intended to constitute an advertisement or medical advice.

SOURCE ASTELLAS PHARMA INC.

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Astellas and Graduate School of Medicine / Faculty of Medicine, Osaka University Enter into Research Collaboration to Develop Pluripotent Stem...

Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and … – Nature.com

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Original post:
Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and ... - Nature.com

iPSC-derived hindbrain organoids to evaluate escitalopram oxalate treatment responses targeting neuropsychiatric … – Nature.com

Reprogramming of PBMCs into iPSCs

iPSC lines were generated as previously described [34, 35]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 3 healthy individuals as well as from 3 AD patients after obtaining informed consent under the oversight of the Johns Hopkins Institutional Review Board. All samples except for AD_2 and AD_3 were obtained through the Johns Hopkins Alzheimers Disease Research Center (ADRC). PBMCs from patients AD_2 and AD_3 are from the ongoing Escitalopram for agitation in Alzheimers disease (S-CitAD) clinical trial (NCT03108846) [33]. PBMCs were expanded in culture, enriched for erythroblasts, and subsequently electroporated for the delivery of episomal vectors MOS, MMK and GBX (Addgene) using a 4DNucleofector (Lonza) according to the manufacturers instructions. After transfection, cells were transferred onto tissue culture plates coated with vitronectin (VTN) in DMEM with 10% FBS (v/v) and supplemented with 5ng/mL of bone morphogenetic protein4 (BMP4). The following day and thereafter, the medium was replaced with xeno-free and feeder-free Essential 8TM medium (E8, ThermoScientific). Between day 13 and 15 of reprogramming, cells presenting the TRA160 pluripotency marker were isolated from the newly generated iPSC colonies using the MACSTM MicroBeads magnetic beads (Miltenyi Biotec). Generated iPSC lines were kept in culture in E8 medium on VTN-coated plates for more than 12 passages before being characterized and used for experiments. For characterization, immunocytochemistry (ICC, see 2.7 below) was performed to check for the presence of multiple pluripotency markers (OCT4, NANOG and TRA-1-60). The iPSC lines underwent flow cytometric analysis to further validate the presence of TRA-1-60 (see 2.4 below).

Human iPSC lines were differentiated into serotonergic (5-HT) neurons by activating WNT and SHH signaling in a 3D in vitro platform [32, 36, 37]. Briefly, to better mimic brain development, the iPSCs were first used to form embryoid bodies (EBs). Induced PSCs were first centrifuged (200 x g, 1min) to form aggregates in ultra-low attachment, round-bottom 96-wells-plates (5000 cells/well, 50L/well) in mTeSRTM medium supplemented with the selective ROCK inhibitor y-27632 (Tocris) on day 0. Starting on the following day, the EBs were cultured to differentiate into neural precursors cells (NPCs) specific to the hindbrain over the course of 3 weeks using serotonergic NPC medium (SNm, see TableS1 in the supplementary information for the full composition). On day 1, 50L of SNm with double the amount of trophic factors were carefully added to start diluting out the mTeSR. On days 2 and 3, 50L of SNm was added to the differentiating EBs. Having reached 200L, 50% (100L) of SNm medium was exchanged daily until day 21. After the 3 initial weeks, growing NPC organoids were transferred to 6-wells-plates (8 NPC-organoids/well, 2mL/well), and they were grown in neural differentiation medium (NDm, see TableS1). NDm was exchanged every 3 days. While in the 6-wells-plates, the organoids were kept on an orbital shaker (ThermoFisher, orbital diameter: 22cm, 50rpm). Hindbrain organoids containing serotonergic neurons (5-HT-organoids) were ready for characterization and experiments after 6 weeks.

In order to evaluate morphological changes of the organoids over time, brightfield images (BF) were taken using an EVOS M5000 microscope (Invitrogen) daily for the first 21 days, then every 3 days until day 42, concurrent with medium changes time points. For the quantification of the area and circularity of the organoids, we developed an in-house algorithm using Python (the full code is available as an open resource on github [38]). Briefly, the images are treated by the code as gray-scale images ranging from 0255 of intensity values. The organoids are segmented using Felzenswalb algorithm [39] with a previous Gaussian smoothing of the images with a 6 pixels size standard deviation kernel. We enforced a minimum size of 3 pixels for the segmentation. In the next step, to improve the results of the segmentation, we manually set a threshold to differentiate background from organoids to 90 (intensity values). Once the segmentation was performed, the code selects the largest region, excluding background, as a binary mask delimiting the organoid. Finally, the area (A) is then computed integrating the pixels inside the mask. To determine the perimeter (P) of the organoid, we computed the integral of the magnitude of the gradient of the binary mask delimiting the organoid [40]. The circularity (C) or roundness of the organoid can be defined from the area and the perimeter as:

$$C=frac{4pi A}{{P}^{2}}$$

(1)

The more round-like the shape, the closest it can approach the maximum of C=1, whereas C values smaller than 1 are indicative of non-circular shapes. The values of the area and perimeter are converted from pixel units to mm using a scale bar given by the microscope, the circularity is adimensional. Representative images of segmentation results are found in FigureS1 (supplementary information).

To evaluate the successful reprogramming of PBMCs into iPSCs, cells were dissociated into singlecell suspensions with TrypLETM (Life Technologies). They were then washed and resuspended in PBS with 1% BSA (wt/v). They were labeled with the primary antibody antihuman TRA160 (Millipore). For the subsequent detection, iPSCs were labeled with secondary antimouse IgMAlexaFluor555 (Thermo Scientific) antibody.

To compare iPSCs and the 5-HT-organoids they were differentiated into, iPSCs and 5-HT-organoids were dissociated with TrypLETM and Gentle cell dissociation reagent (STEM cells technologies) respectively. Cells were washed and resuspended in PBS with 1% BSA (wt/v), following which they were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciencence) according to manufacturers instructions. Samples were subsequently labeled using AlexaFluor488 conjugated anti-human TUJ1 and AlexaFluor647 conjugated anti-human TPH2 antibodies (ThermoScientific). The former is a general neuronal marker, whereas the latter is specific for serotonergic neurons.

All samples were analyzed on a BD LRS Fortessa (BD Biosciences) or on a SH800S cell sorter (SONY Biotechnology). The data was processed using FlowJoTM v10.8.1 software. A full list of the antibodies used for flow cytometry and ICC is available in TableS2 in the SI.

To evaluate the differentiation of iPSCs into 5-HT-organoids, quantitative reverse transcription PCR (qRT-PCR) analysis was performed. Messenger RNA (mRNA) was extracted from cellular pellets of iPSCs and 5-HT-organoids using RNA extraction kit (Zymo research), and it was transcribed into complementary DNA (cDNA) by reverse transcriptase using the Superscript III kit (Invitrogen) following manufacturers instructions. The generated cDNAs were used as the template for the qPCR reaction with iTaq Universal SYBR Green (Biorad), which was performed with a CFX Connect thermal cycler (Biorad). The primers used were obtained from Integrated DNA technologies and they were for TRA-1-60 (iPSC marker), NKX2.2 (serotonergic NPC), LMXbI and TUJ1 (neurons), TPH2 and FEV (serotonergic neurons). All forward and reverse primer sequences (purchased from Integrated DNA Technologies) are listed in TableS3 (SI).

Hindbrain organoids were washed three times with D-PBS (pH 7.4) and placed in a 1.5mL centrifugation tube with 1.2mL of freshly prepared 4% (v/v) paraformaldehyde and left incubating for 18h at 4C. They were then washed for 10min with D-PBS with 0.1% (v/v) Tween20 (Sigma) 3 times. For cryoprotection, the organoids were placed in 30% (wt/v) sucrose in D-PBS and left to equilibrate at 4C until they did not float in it anymore (ca. 4h, but it can vary depending on organoid size and density). The organoids were then transferred to an embedding mold which was carefully filled with O.C.T. compound embedding matrix (ThermoFisher). Snap freezing was done by submerging the molds with the embedded organoids in a slurry of dry ice added to 96% ethanol. The frozen organoids were then stored at 80C before being sectioned in 10m slices at the Johns Hopkins University SOM Microscopy facility.

Evaluation of pluripotency markers by ICC on adherent human iPSCs was performed as previously described [35]. Briefly, adherent iPSCs in 12-well plates were washed in PBS and fixed with 4% (v/v) paraformaldehyde in PBS (pH 7.4) for 15min, and permeabilized with Triton X-100 (0.1%, v/v in PBS). To limit non-specific binding, cells were blocked in 10% goat serum (v/v in PBS) for 1h at 4C. They were then stained with either one of the primary antibodies for pluripotency markers, i.e., anti-human TRA-1-60, NANOG andOCT4 at 4Covernight. Cells were subsequently washed with PBS, and they were then incubated with the appropriate secondary antibody for 1h at 4C. In the final step, cells were washed with PBS three times, and then stained with DAPI to visualize the nuclei.

Cryo-preserved and sectioned 5-HT-organoids were similarly stained for ICC to confirm the presence of neuronal marker TUJ1, serotonin (5-HT), and neural progenitor cells (NPCs) markers Nestin and NKX2.2 (necessary to determine serotonergic fate) [41]. Confocal fluorescence imaging was performed with a Leica SP8 inverted microscope (DMi8CEL), and the images were analyzed with a Leica LAS X software.

A full list of the antibodies used for flow cytometry and ICC is available in TableS3 in the SI.

Levels of 5-HT present in the extracellular supernatant were measured by enzyme-linked immunosorbent assay (ELISA) using the Serotonin ELISA kit (Enzo Life Sciences) according to the manufacturers instructions. To test the effect of the SSRI escitalopram oxalate, 10 and 100M of the drug were added to NDm and incubated with the eight 5-HT organoids for 1h prior to repeat measurement of supernatant 5-HT. The concentration range was initially chosen based on prior literature [36]; a metabolic activity assay was performed to ensure that the used concentrations were not toxic in our systems (see FigureS3 in the supplementary information).

All experiments were performed in at least 3 biologically independent replicates (n), and at least 36 technical repeats (N) unless stated otherwise. The results are presented as meanstandard deviation (SD). One-way ANOVA test, followed by Tukeys Honest Significant Difference test, was performed to pairwise evaluate if there were statistically significant mean differences between groups for Fig.6bd. The results were displayed using GraphPad Prism version 9.0.0 (121) for Windows, GraphPad Software, San Diego, California USA, http://www.graphpad.com. Statistically significant results are indicated with their respective p-values and asterisks as follows: p0.05 (*), p0.01 (**) or p0.001 (***).

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iPSC-derived hindbrain organoids to evaluate escitalopram oxalate treatment responses targeting neuropsychiatric ... - Nature.com

Shinobi and Anocca to advance cancer killing iPS-T cell therapies – BioProcess Insider

Under the terms of the agreement, Shinobi will combine its iPS-T cell platform with Anoccas TCR discovery platform to create a new class of TCR-iPS-Ts.

Anoccas platform allows the scale-out of TCR-T development and delivers libraries of clinical candidate TCRs that span multiple solid tumor cancer targets across broad patient segments, Reagan Jarvis, CEO of Anocca told BioProcess Insider.

By combining Anocca TCRs with the Shinobi Katana platform, we envision a rapid, efficient, novel and transformative product manufacture modality. Under this antigenic targets on a patients tumor are matched with Anoccas TCR library and introduced in a plug-and-play' manner into clinic-ready Shinobi iPS-T-cells. We anticipate delivering initial validatory data within the first year of the partnership and this will form the springboard for further validation and product development over the coming years.

Anocca TCR platform is designed to recreate human T-cell biology in the lab to precisely map T-cell targets and identify highly specific and potent TCRs. The platform uses advanced tests with programmable human cells to carefully analyze and find real disease targets and the specific T-cell receptors that recognize them, according to the company.

Meanwhile, Shinobis Katana platform is said to enable rapid pipeline creation by driving iPS-T cell differentiation without defining antigen specificity. This allows the development of an immune evasive CD8ab iPS-T cell platform that can then be armed with any receptor. CD8ab iPS-T cells are critical class I-restricted T cells responsible for killing cancerous or virally infected cells and mediating adaptive immunity.

Our Katana technology allows us to have a fully engineered CD8ab iPS-T cell which can then be modified to efficiently introduce a CAR or TCR in a plug-and-play manner, Dan Kemp, CEO of Shinobi told us.

Key challenges in the development of off-the-shelf TCR-iPS-T cell therapies are production and robustness of the process to differentiate the cell phenotype and the ability to produce cells at scale. From Anoccas perspective, we have been impressed by the Shinobi platform and its potential to deliver against these criteria. Our partnership is aimed at addressing these key challenges in a stepwise manner.

The financials of this partnership have not been disclosed.

Original post:
Shinobi and Anocca to advance cancer killing iPS-T cell therapies - BioProcess Insider

Improving Models to Study the Human Heart – News Center – Feinberg News Center

Northwestern Medicine scientists have developed a new method to measure and optimize the maturation process of cultured heart muscle cells, an approach that has the potential to set the future standard for a common cell model in scientific research, according to details published in Cell Reports.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are cultured heart muscle cell models widely used to study a variety of human heart disease and responses to experimental drugs. However, newly cultured cardiomyocytes dont accurately reflect mature heart muscle cells in adult humans, and previous methods of measuring maturation were not high throughput.

Differences in cellular maturity may affect the results of various experiments and studies done using the cells, so understanding when the cells are suitable is critical, said Paul Burridge, PhD, associate professor of Pharmacology and senior author of the study.

The hiPSC-CM models dont perfectly match an adult cardiomyocyte, said Burridge, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University. There are a number of ways you can make these cardiomyocytes more mature, but all of those techniques are time-consuming and not always compatible with the assays we perform, so we were really interested in what we could do to make these cardiomyocytes match an adult cardiomyocyte as much as possible.

In the study, Burridge and his collaborators cultured hiPSC-CMs and performed high-throughput assays to measure maturation of the cells. They found several factors could indicate when the cells are mature, including gene expression, mitochondrial function and electrical activity.

Building off this discovery, the investigators then developed cellular media combinations of compounds and nutrients designed to support cultured cellular growth and optimized it for the rapid maturation of the heart muscle cells.

The new measurement method and optimized cellular media will make it easier for scientists to study human heart cells, Burridge said.

IPS cell-derived cardiomyocytes appear to be one of the most powerful applications of the IPS cell technology in drug screening, Burridge said. The cells basically represent the heart cells of a patient. Whether were interested in the effects of drugs, arrhythmia, or heart failure, we want to have the best models possible. Here, we have improved the fidelity of that model without making it more complex.

Moving forward, Burridge and his collaborators will continue to optimize the model to match human heart muscle cells as closely as possible, he said, and potentially reduce the need for animal models in scientific research.

The better we can make this, the more work can be done in this cell culture model rather than in animal models such as mice, as it was done in the past, Burridge said. By improving the quality of this model, thats going to get us a little bit closer.

The study was supported by National Institutes of Health grants R01 CA220002 and CA261898 as well as funding from the Leducq Foundation.

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Improving Models to Study the Human Heart - News Center - Feinberg News Center

In vivo cyclic overexpression of Yamanaka factors restricted to neurons reverses age-associated phenotypes and … – Nature.com

Characterization of CamKII-transgenic mice: neuron-restricted reprogramming in young mice

Since the transgenic model we used is entirely novel, we aimed to characterize it initially and verify that indeed Yamanaka factors (YF) were being effectively expressed. To do this, we began by studying the first offspring whose genotyping confirmed the presence of both transgenes. We maintained continuous activation of the doxycycline-inducible system from birth until the mice reached three months of age (when the nervous system should be fully formed), time when we collected samples.

To assure that exogenous YF were effectively expressed, quantitative PCR (qPCR) was performed against the sequence E2A-cMyc of the transgenes OSKM using hippocampus, neocortex and cerebellum samples. The results revealed a clearly higher expression in -CaMKII-OSKM compared to transgenic control mice in hippocampus and neocortex, but no difference was observed in the cerebellum between transgenic and control mice, being almost undetectable (Supplementary Fig.1a).

Attending the expression of one of the YFs (Klf4) in cerebral histological regions, we were able to determine more precisely the specific areas in which the promoters were active throughout the experiment (Supplementary Fig.1b). It is important to note that due to the nature of the transgene, the expression of Klf4 (located in the third position within the gene order of the construct, after Oct4 and Sox2) implies that the other genes should have also been expressed in the neuron, so the expression pattern observed with Klf4 would correspond to that of the transgene as a whole.

The highest expression occurs in different neuronal layers of the hippocampal formation (DG, CA3, CA1, Subiculum), dorsal and ventral striatum, and neocortex (somatosensory, somatomotor, visual or orbital areas). Some expression was also observed in the thalamus but at a much lower level. No expression was found in other diencephalic regions, such as the globus pallidus, or in brainstem regions such as the substantia nigra, superior or inferior colliculus, cerebellum, or medulla. The varied pattern of -CaMKII promoter expression throughout the different cerebral regions in these mice is in line with that described previously20.

In order to better understand the molecular process behind, we have focused first on the hippocampal formation, which not only have exhibited one of the highest Klf4 protein expressions in this model but also because this region is involved in different key brain processes such as memory functioning or adult neurogenesis, both processes affected by ageing21. We obtained bulk transcriptomic data from the hippocampi of 8 different animals to which doxycycline was not administered since birth, allowing neuronal cells from double transgenic mice to express YF continuously since birth. RNA-seq analysis (ENA accession number is PRJEB56610), by using Deseq2722 R software package, detected 1419 differentially expressed genes (DEG) (q value<0.05) in neuronal-reprogrammed animals regarding control mice. From all of them, 604 DEG resulted in decreased and 815 in increased expression. The MA-plot shows the log2 fold changes (M) between two conditions over the mean of normalised counts (A) for all samples (Supplementary Fig.1c). In the heatmap (Supplementary Fig.1d), is also shown how the expression of YF in a subpopulation of neurons, has led to significant changes in the transcriptome of a relevant group of genes. According to the Gene Ontology (GO) knowledgebase, which is the largest source of information on the functions of genes and proteins, and to the Kyoto Encyclopedia of Genes and Genomes (KEGG), we have been able to track down groups of differentially expressed genes (DEG) involved in certain cellular functions (Supplementary Fig.1e-f; Supplementary Data1). Data obtained revealed changes in the expression of genes related with regulation of nervous system development (46 DEG; 1.68E-07 p.adj), stem cell differentiation (21 DEG; 0.027 p.adj) or maintenance (17 DEG; 0.02 p.adj), central nervous system neuron differentiation (23 DEG; 1.74E-03 p.adj) or specifically in regulation of neuron differentiation (31 DEG; 1.91E-05 p.adj), would confirm that processes related with reprogramming in -CaMKII-OSKM mice are taken place. In addition, transcription changes were found in genes related with: extracellular matrix organization (48 DEG: 5.09E-10 p.adj), structure (29 DEG; 1.29E-08 p.adj) or in the regulation of cell-cell adhesion (62 DEG; 2.86E-10 p.adj); in the regulation of synaptic organization (38 DEG; 1.68E-07 p.adj) and of synaptic structure or activity (39 DEG; 1.20E-07 p.adj); learning or memory (39 DEG; 4.30E-06 p.adj) and cognition (42 DEG; 3.85E-06 p.adj); dendrite morphology (26 DEG; 3.26E-05 p.adj) or development (34 DEG; 0.00019 p.adj), axogenesis (61 DEG; 3.07E-09 p.adj) as well as in regulation of neurogenesis (57 DEG; 6.93E-09 p.adj). It is important to note that all these functions are ultimately affected with ageing.

Furthermore, using a bioinformatics tool such as Ingenuity Pathway Analysis (IPA)23, we were able to identify a common upstream regulator of downstream genes. IPA revealed several different types of upstream molecules (~400), including transcription regulators, transporters, cytokines, growth factors, kinases or various enzymes. Among all upstream regulators, HMG20A, IL4, FGFR, C1QA, KTMT2D, KAT2A or CREBBP are within the top 20 most significantly activated regulators (P<10-05 and Z-score 2; among the top 30 upstream regulator). These regulators have been associated with epigenetic modifications (KTMT2D, KAT2A), neuronal differentiation (HMG20A, FGFR, KTMT2D), neurodevelopment (C1QA, FGFR, CREBBP), the ageing process (IL4, FGFR) or memory functioning (KAT2A). On the other hand, APOE, TP63, Ptprd or PSEN1 were among the most significantly inhibited regulators (P<10-04 and Z-score 2; among the top 30 upstream regulator), and they are also involved in ageing or ageing-associated diseases (APOE, TP63, Ptprd or PSEN1), cell adhesion (Ptprd) or neural differentiation (PSEN1).

During these experiments the impact of continuous neuron-restricted reprogramming on mortality rate was analysed. Thus, in the case of these young mice that underwent continuous induction of transgenes from birth, a high mortality rate (around 60%) was observed, along with the presence of hydrocephalus in some cases. However, for the mice in which the transgene system was only activated in adulthood at 6 months old, whose study will be described below, the mortality rate decreased to zero. Neither in the case of continuous induction nor in the case of cyclic induction did we find teratomas, confirming that the expression of YF in neurons did not lead to cellular dedifferentiation in vivo. This result is in line with the findings of Kim and colleagues17.

Next, we proceed to study the impact of neuron-restricted reprogramming on adult mice during long-term treatment, initiating the treatment in mice aged 6 months to nearly one-year-old, a protocol similar to that described previously13. Some mice were subjected to continuous factor expression by the continuous withdrawal of doxycycline over the 4-month treatment period, while another group of mice underwent cyclic doxycycline administration. This cyclic protocol involved administering water for 3 days per week and doxycycline for the remaining 4 days (Fig.1). The cyclical expression of Yamanaka factors was confirmed by the histological study of Klf4 protein expression at different time intervals during a week cyclical protocol (Supplementary Fig.2a-b). The analysis of immunofluorescence obtained from Klf4 Yamanaka factor expression have shown that indeed, after 3 days of induction, there is a significant increase (p value=0.0043) in the expression of Klf4 Yamanaka factor (Supplementary Fig.2c). This induction returns to day 0 levels after 4 days of continuous doxycycline administration (p value=0.0146).

a Crossbreeding was conducted between -CaMKII-tTA and TetO-OSKM transgenic mice to generate the -CaMKII-OSKM mice. b Schematic representation of the OSKM transgene showing the location of DNA sequences encoding 2A peptides that separate each Yamanaka factor. c Temporal representation of the three OSKM treatments applied to the murine model in the present study. Doxycycline administration prevents binding between the transactivator tTA and the TetO promoter and thus inhibits transcription of Yamanaka factors.

To characterize the histological expression of YF in adult -CaMKII mice, we employed antibodies targeting KLF4, just as in the previously shown study with young mice (Fig.2a, b; Supplementary Fig.3a). Quantitative analysis of immunohistofluorescence in adult -CaMKII mice subjected to cyclic transgene induction from 6 to 11 months, demonstrated significantly higher YF expression compared to transgenic control mice (Fig.2c, d). This difference was more pronounced in the deeper layers of the somatosensory neocortex (Fig.2c). In the control group, YF expression was nearly absent in all regions of the cerebral cortex, as shown in Fig.2a. Additionally, continuous transgene expression resulted in higher YF expression levels compared to transgenic control mice and adult -CaMKII mice with cyclic YF expression. This difference was more evident in the hippocampal region (Supplementary Fig.3b, c).

a, b Representative microphotographs of the Klf4 Yamanaka Factor immunoreactivity (in green) obtained in sagittal brain sections from control 11 months old adult mice (n=5) (a), -CaMKII-OSKM mice with cyclic induction (n=5) of transgene system from 6 to 11 months of age (b). Schematic outlines approximating the boundaries of some of the most relevant areas of the central nervous system have been overlaid on the microphotographs. Scale bar shown in a and b indicates 1000 m. c, d Graphical representation of the meanSEM of the percentage of occupied area by Klf4-immunoreactivity in somatosensory neocortex, distinguishing deep layers from superficial (c) and different regions from hippocampal region, including CA1, CA3 and dentate gyrus (d). *p<0.05 and ** p<0.01 by Students paired t-test.

With respect to the regional distribution of Klf4 expression, adult -CaMKII mice with cyclic and continuous expression of YF exhibited an immunofluorescence pattern of Klf4 protein expression similar to that of mice with continuous expression from birth at 3 months of age (Fig.2b; Supplementary Fig.3a). The highest expression was found throughout all the neocortex, mainly in deeper layers. Expression was also found in the hippocampus, subiculum, caudate putamen, piriform cortex and thalamus, but cerebellum or other medullary nucleus seemed to lack YF expression.

We first analysed the animals anxiety levels by observing their exploration behaviour within the central area of the box. Typically, rodents tend to remain close to the walls and avoid open spaces, a behaviour known as thigmotaxis24. As rodents age, it appears that they tend to spend less time in the central zone of the open field, which would translate to higher levels of anxiety25,26. The results showed statistically significant increase (p value=0.0003) in the time spent by the -CaMKII-OSKM mouse in the central zone of the tray when they had cyclic treatment in comparison with the control group (Fig.3a, b). As already described (see Materials and Methods), a short-term (2hours) novel object recognition test was used to assess memory performance. The results of the test (Fig.3c, d) showed a higher memory index in terms of time (P value=0,0149) and entries (P value=0,0204) in -CaMKII-OSKM regarding the control group. Spatial memory was also evaluated through the Y-maze test (Fig.3e, f), which showed improvements in the -CaMKII-OSKM mice with cyclical administration of doxycycline with respect to the control group (P value=0,0422). Therefore, cyclic activation of YF expression restricted to a subpopulation of neurons was enough to improve different types of memory in middle-age mice. Contrary to the cognitive effects found in -CaMKII-OSKM adult mice with the cyclical induction of YF, continuous induction did not result in significant changes, either in the open field test or in the other tests conducted.

a, c, e Schematic representation of the organisation of the space where the behavioural tests were performed, a Open field test, b Novel Object Recognition test and c Y maze test. Representative tracking maps obtained with Any Maze software, showing the trajectory of the centre of the rodent during the behavioural test. b, d, f Graphical representation of the meanSEM of the time (s) between the number of entries in the area analyzed (see Material & Methods) in different experimental groups. Control 11 months old adult mice (n=8) and -CaMKII-OSKM mice with cyclic induction (n=9); control 11 months old adult mice (n=8) and -CaMKII-OSKM mice with continuous induction (n=7). *p<0.05, ** p<0.01, *** p<0.001.

Given that only the cyclical induction of YF in adult neurons yielded noteworthy improvements in cognition compared to continuous induction, we opted to focus on this approach, which would entail expression of YF within the -CamKII promoter subpopulation of neurons.

We obtained transcriptomic data from both hippocampal and neocortical tissues of adult animals. Bulk RNA-seq analysis (ENA accession number is PRJEB65922), by using Deseq2722 R software package, detected in total 94 differentially expressed genes (DEG) (q value<0.05) in neuronal-reprogrammed animals regarding control mice (Supplementary Data2). Out of all of them, the majority (~75%) showed decreased expression (70 DEG), while around 25% their expression was found to have increased. The gene expression data can be visualized in Fig.4 where the MA-plot is shown for all samples in the neocortex and in the hippocampus (Fig.4a, b; Supplementary Data2). In the heatmaps (Fig.4c, d), it is also shown how the expression of YF in a subpopulation of neurons, has led to significant changes in the transcriptome of a relevant group of genes.

a, b MA-plot from neocortex (a) and hippocampus (b) samples which represent genes coloured in blue that have q values less than 0.05. Points which fall out of the window are plotted as open triangles pointing either up or down. Heatmap from neocortex (c) and hippocampus (d) samples. Data are displayed in a grid where each row corresponds to a gene and each column to a sample (from two different conditions). The colour and intensity in the heatmap represent changes of gene expression from the list of genes with q value in the Principal Component Analysis (PCA). Emapplot from neocortex (e) and hippocampal (f) samples, showing an enrichment Map for enrichment result of over-representation test or gene set enrichment analysis. g GO term analysis (cellular component) of altered genes.

According to the Gene Ontology (GO) knowledgebase, we have been able to track down groups of differentially expressed genes (DEG) involved in certain cellular functions (Fig.4e, f; raw data in Supplementary Data2). Firstly, it is important to note that, when examining the cellular component of ORA analysis (considering the both hippocampus and neocortex), the transcriptomic data revealed that reported changes were mainly located in specific neuron compartments, with the dendritic component standing out prominently (Fig.4g). This finding confirms the specificity of YF expression solely in neurons. Data obtained revealed that genes with altered expression (qvalue<0.05) were included in different biological processes, as regulation of nervous system development (Nectin3/Chrna4/Lrrtm4/Gabra5; 3.7E-04 padj) and neuron differentiation (Dab1/Trpc6/Brinp3/Neurog2; 5.5E-04 padj) that indicate functions compatible with a reprogramming process. Moreover, in general many of these differentially expressed genes (DEG), could be primarily grouped into alterations in processes related to the extracellular matrix (e.g. ECM organization Itga8/Col15a1/Adamts16/Fbln2/Grem1/Col22a1; 5.45E-04 Padj) and cell adhesion (e.g. regulation of cell substrate-adhesion; Col26a1/Pcsk5/Thy1/Ajap1/Fbln2/Ppm1f/Grem1, 7.27E-06), neuronal activity involving different classes of neurotransmitters (13 different processes, e.g., neurotransmitter receptor activity; Chrna4/Chrnb3/Gabra5/Htr2a/Hrh3, 2.96E-05 padj), cognition (Itga8/Chrna4/Gabra5/Htr2a/Hrh3/Jun, 9.4E-04), and processes always associated with neuronal structures and functions, with a particular emphasis on postsynaptic processes (e.g, postsynaptic specialization; Nectin3/Itga8/Chrna4/Dab1/Lrrtm4/Gabra5/Als2/Lzts3, 9.53E-05 padj).

Taking into account the hippocampus and the neocortex separately, the expression of these factors seems to have led to a somewhat more intense reprogramming process in the second region compared to the hippocampal region, considering the number of genes with altered expression (43 DEGs in the hippocampus vs. 59 DEGs in the neocortex). Among the genes with the most statistically significant differential expression, notable examples relate to the extracellular matrix, with collagen alpha 1 type XXVI (Col26a1; 7.5831E-11 padj) in the hippocampus and collagen alpha 1 type XXII (Col22a1; 1.63E-4 padj) in the neocortex. Furthermore, zinc finger proteins, like Zfp804b (3.6816E-06 padj) in the neocortex and Zfp386 (4.94E-09 padj) in the hippocampus, are noteworthy. Given their capacity to bind to chromatin, these proteins are believed to play a central role in neuronal reprogramming processes27 and the latter have been involved in silencing LINE-1 elements28,29. In addition, there have been genes whose expression has been found to be altered in both cortical areas, such as Glis3 (another zinc finger protein) and Fbln2. The expression of the Glis3 gene, functionally involved in reprogramming processes30 and known to increase with aging31,32, was significantly reduced in both hippocampal and neocortical regions in this study. Fbln2 is an extracellular matrix protein with roles in tissue remodelling and embryonic development33,34.

Since transcriptomic studies have revealed significant effects of partial reprogramming on regulatory neuronal activity genes we aimed to investigate, in a more specific manner, how these changes in neuronal activity may have contributed to the cognitive improvement observed in these animals during partial reprogramming. For this purpose, we conducted histological analyses on tissue samples using an immediate early gene c-Fos marker. In neurons, c-Fos expression is induced under conditions of neuronal plasticity, including learning and memory35. It has been widely used as a neuronal activity marker since they are rapidly and transiently induced by neuronal stimuli in the brain36. In this study, the mice were immediately perfused upon completion of the memory test. In this way, we were able to study the activity levels of the memory circuits during the execution of these memory tests. The analysis of c-Fos-immunoreactive cells was focused on the hippocampus (Fig.5a), due to its essential role on recognition/spatial memory performance. We found a higher number of neurons active (c-Fos-immunoreactive) just after memory test performance in -CaMKII-OSKM transgenic mice regarding transgenic control mice, in both granular cell layer of dentate gyrus and in the pyramidal cell layer of CA1 (Fig.5b). This result potentially indicates more active circuits during memory testing due to YF expression restricted to neurons.

a Representative microphotographs of immunoreactivity obtained for c-Fos protein expression (in red) in the hippocampus region from control (n=8) and -CaMKII-OSKM mice (n=10). Scale bar indicates 200 m. b Graphical representation of the meanSEM of c-Fos-immunoreactive cells per mm3 at different neuronal layers of the hippocampal region (Dentate gyrus, DG; CA1; CA3). *p<0.05.

Additionally, we have found in these mice a significant inverse correlation (R=0.964) between levels of Klf4 expression and density of c-Fos-immunoreactive cells (Supplementary Fig.4). Excessive expression of Klf4 led to lower increase of c-Fos-immunoreactive cell density in the hippocampus of -CaMKII-OSKM transgenic mice. These results are consistent with those found in -CaMKII-OSKM mice with continuous induction of the YF. In these mice, where Klf4-YF expression is continuous, we observed worse cognitive performance compared to those with cyclical induction. All these results underscore once again the importance of the level of induction of the YF. Moderate rather than excessive induction is what achieves beneficial effects on the cognition of aged mice.

Considering significant changes previously identified in the extracellular matrix (ECM) as a result of reprogramming in young -CaMKII-OSKM mice (Supplementary Fig.1, Supplementary Fig.5), which led to an overall reduction in its structure, in addition to transcriptomic data obtained from -CaMKII-OSKM adult mice showing significant alterations in genes related to ECM, we aimed to investigate whether partial reprogramming in adult mice would result in youthful ECM reorganization. Thus, we studied the expression of the cartilage-specific core protein proteoglycan (aggrecan), which binds to specific proteoglycans and allows visualisation of the so-called perineuronal extracellular matrix networks (Fig.6a and d from the overall panoramic view). Immunoreactivity analysis for this protein was carried out in both the neocortex and hippocampal formation at both experimental groups (control and -CamK-OSKM adult mice with cyclic overexpression of YF). Figure6a shows representative microphotographs of the neocortex using an antibody against aggrecan protein, where, to facilitate the analysis, the supragranular layers (layers I-IV) have been distinguished from the infragranular layers (layers V and VI). The results have shown a prominent inclination towards an overall reduction in the percentage of area occupied by the aggrecan protein across the entire neocortex (p value=0.0518), attaining statistical significance within the deeper neocortical layers (V and VI) among -CaMKII-OSKM mice in comparison to the control group (p value=0.0258; Fig.6b).

a Representative microphotographs of immunoreactivity obtained for Aggrecan protein expression (in red) in the somatosensorial neocortex from control (n=8) and -CaMKII-OSKM mice (n=9). On the right side of the panel are enlargements of the panoramic view displaying the structure of perineuronal networks formed by the extracellular matrix. Scale bar shown in A indicates 200 m and 15 m in the magnification. b Graphical representation of the meanSEM of the percentage of area occupied by Aggrecan-immunoreactive cells per mm3 in total volume of the somatosensory neocortical region and at different cortical layers. *p<0.05. c Density of perineuronal net units (PNNs) in the somatosensorial neocortex (number of Aggrecan-immunoreactive PNNs in each brain slice by the volume of the somatosensory neocortical region, *p<0.05). d Representative microphotographs of immunoreactivity obtained for Aggrecan protein expression (in red) in the hippocampus of the different murine models. On the right side of the panel are enlargements of the panoramic view displaying the structure of perineuronal networks formed by the extracellular matrix. Scale bar shown in (a) indicates 200 m and 15 m in the magnification. e Representation of the meanSEM of the percentage of area occupied by Aggrecan immunoreactive per mm3 at different neuronal layers of the hippocampal region (Dentate gyrus, DG; CA1; CA3). f Density of perineuronal net units (PNNs) in total hippocampus (number of Aggrecan-immunoreactive PNNs in each brain slice by the volume of the area analysed).

In line with these results, the analysis of the density of perineuronal net units also shows a significant reduction following the induction of partial reprogramming in -CaMKII-OSKM mice (Fig.6c). In contrast to the neocortical areas, noteworthy statistical differences were not found in the hippocampus between both experimental groups for the area occupied by the immunoreactive aggrecan matrix (Fig.6e), nor regarding the density of PNN units (Fig.6f). This general reduction found by immunofluorescence detection in aggrecan-immunoreactive extracellular matrix has been corroborated by Western blot technique (Supplementary Fig.6a, b). We observed a highly significant decrease (P=0.0001) in its expression following the cyclical induction of the Yamanaka factors. These data confirm the significant role of extracellular matrix reorganization during cyclical reprogramming processes in the brain.

In vitro studies have demonstrated that YF alone is not sufficient to induce neuronal dedifferentiation17. In this study, we decided to try to validate these findings in vivo, determining whether, under YF expression, neurons could undergo dedifferentiation. For this analysis, we employed a distinct set of antibodies. Doublecortin protein (Dcx) is expressed in migrating neuroblasts and immature neurons, making it a reliable marker for adult neurogenesis. In general, in adult rodents, it is only possible to find immature neurons in regions where neurogenesis occurs, which are typically only two, one of which is the subgranular zone (SGZ) of the hippocampal dentate gyrus. Thus, we tried to detect doublecortin labelling outside of the subgranular zone in -CaMKII-OSKM mice. This would indicate that processes of dedifferentiation owing to YF expression from mature neurons to a previous state of maturation have occurred. In these animals, throughout the cerebral cortex we only observed doublecortin labelling in the subgranular zone, similar to what was seen in the control group. We did not find these cells in the rest of the hippocampus or the neocortex. Moreover, we observed no differences in the density of Dcx-immunoreactive cells between the control and -CamKII-OSKM mice in SGZ (Supplementary Fig.7a, d). Furthermore, we aimed to study other markers of earlier neuronal development such as the intermediate progenitor marker T-box brain gene 2 (Tbr2) to ensure that partial reprogramming was not regressing to even earlier stages than those identified by the doublecortin marker. The results in the SGZ revealed no differences in Tbr2 expression between the transgenic and control groups (Supplementary Fig.7b, e). Additionally, 5-chloro-2-deoxyuridine (CldU) was administered three weeks before perfusion for each mouse in both experimental groups to identify cells that were newly generated at that time. Results have shown how the number of three weeks-old cells labelled with CldU was not significantly changed in the DG of the -CaMKII-OSKM, indicating that partial reprogramming by YF not only does it not appear to influence adult neurogenesis itself, but it also would not affect the proliferation of new cells in adult mice (Supplementary Fig.7c). Differences in the density of CldU-labelled cells between control and -CaMKII-OSKM adult mice in the somatosensory neocortex were not found either (Supplementary Fig.7c, f).

Considering that the reprogramming induced by Yamanaka factors correlates with epigenetic changes7,37, in this study we aimed to verify whether the selective partial reprogramming of neurons led them to more youthful epigenetic states. Early studies in rats showed that methylation of histones H3 and H4 changes gradually with increasing age38. Moreover, a systematic study of posttranslational modifications of histones in the brain of senescence-accelerated prone mouse 8 (SAMP8) model revealed a significant decrease of H4K20me3 marker during ageing39. Here, we have found a remethylation of this epigenetic marker at H4 after cyclic neuronal induction of YF expression in adult mice (Fig.7). The results showed that H4K20me3 (histone 4 lysine 20 trimethylation) marker increases in -CamKII-OSKM adult mice overexpressing YF in a cyclic manner regarding control mice throughout all the neocortex layers (p value=0.0124; Fig.7a, b), as well as in CA1 (p value=0.0372) and CA3 (p value=0.0288) pyramidal cell layers at the hippocampal region (Fig.7c, d), but not in the granular cell layer of DG. According to previous studies these epigenetic changes in areas where partial reprogramming is taking out would lead to a more youthful epigenetic pattern in those reprogrammed cortical neurons39.

a Representative microphotographs of immunoreactivity obtained for H4K20me3 expression marker (in green) in the somatosensory neocortex from control (n=8) and -CaMKII-OSKM mice (n=9). On the right side of the panel, enlargements are displayed, showing H4K20me3-immunoreactivity expression in a group of cells present in the deeper neocortical layers. Scale bar shown indicates 200 m and 15 m in the magnification. b Representative percentage of mean intensity (arbitrary units) obtained from H4K20me3 immunoreactivity in neocortex, distinguishing supragranular layer (I-IV) and infragranular layers (V-VI). *p<0.05. c Representative microphotographs of immunoreactivity obtained for H4K20me3 expression marker (in green) in the hippocampal region of the different murine models. On the right side of the panel, enlargements are displayed, showing H4K20me3-immunoreactivity expression in a group of cells present in the pyramidal neuronal layer of the CA1 region. Scale bar shown indicates 200 m and 15 m in the magnification. d Percentage of mean intensity (arbitrary units) obtained from H4K20me3 immunoreactivity in hippocampus, distinguishing CA1, CA3 and dentate gyrus (DG). *p<0.05.

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Global Induced Pluripotent Stem Cells Production Market, By End User

Pharmaceutical & biotechnology companies Research & academic institutes Contract research organizations

Browse Full Report: https://www.infinitivedataexpert.com/industry-report/induced-pluripotent-stem-cells-production-market

Regional Segmentation of the Global Induced Pluripotent Stem Cells Production Market

North America (the United States, Canada, and Mexico) Asia-Pacific (China, Japan, Korea, India, and Southeast Asia) Europe (Germany, France, UK, Russia, and Italy) The Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, and South Africa) South America (Brazil, Argentina, Colombia, etc.)

Responses that the report accepts:

The size of the market and its growth rate over the next few years. The main things that drive the Induced Pluripotent Stem Cells Production Market. Key market trends that are making the Induced Pluripotent Stem Cells Production Market grow faster. Threats to the growth of the market. Key sellers of Induced Pluripotent Stem Cells Production Market. SWOT study in depth. The chances and risks that the current sellers in the Global Induced Pluripotent Stem Cells Production Market face. Trending factors that affect the market in different parts of the world. Strategic efforts are centred on the top vendors. A PEST study of the market in the five most important areas.

Contact Info

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About Us

Infinitive Data Expert is a leading distributor of market research report with more than 600+ global clients. As a market research company, we take pride in equipping our clients with insights and data that holds the power to truly make a difference to their business. Our mission is singular and well-defined - we want to help our clients envisage their business environment so that they are able to make informed, strategic and therefore successful decisions for themselves.

This release was published on openPR.

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Induced Pluripotent Stem Cells Production Market Will Hit Big - openPR