Tag Archives: pluripotent stem cells (hPSCs)

Protocols for mouse-human chimeric embryos

This work on a type of species boundary-crossing could be very disturbing for some folks. That said, here’s more about the science from a July 2, 2021 news item on phys.org,

A year after University at Buffalo [in New York state] scientists demonstrated that it was possible to produce millions of mature human cells in a mouse embryo, they have published a detailed description of the method so that other laboratories can do it, too.

A July 2, 2021 University at Buffalo (UB) news release (also on EurekAlert) by Ellen Goldbaum, which originated the news item, explains why scientists have created these chimeras,

The ability to produce millions of mature human cells in a living organism, called a chimera, which contains the cells of two species, is critical if the ultimate promise of stem cells to treat or cure human disease is to be realized. But to produce those mature cells, human primed stem cells must be converted back into an earlier, less developed naive state so that the human stem cells can co-develop with the inner cell mass in a mouse blastocyst.

The protocol outlining how to do that has now been published in Nature Protocols by the UB scientists. They were invited to publish it because of the significant interest generated by the team’s initial publication describing their breakthrough last May [2020].

“This paper will enable many scientists to use this new platform to study the human disease of their interest,” said Jian Feng, PhD, professor of physiology and biophysics in the Jacobs School of Medicine and Biomedical Sciences at UB and senior author. “Over time, it will transform biomedical research toward a more effective use of the human model system to directly study virtually any inborn condition of an individual. It will stimulate unforeseen discoveries and applications that may fundamentally change our understanding of human biology and medicine.”

The protocol will allow scientists to create animal models that Feng said provide a much more realistic picture of embryonic development than has ever been possible. These more realistic animal models also will have the potential to reveal the mechaniswms behind numerous diseases, especially those that afflict individuals from birth.

Better mouse models

“This step-by-step protocol will benefit the entire field by enabling other scientists to use our methods to generate chimeras to study human diseases that they are experts in,” said Feng. “It will lead to the generation of better mouse models for various human diseases, such as sickle cell anemia, COVID-19 and many others, or various human developmental disorders.” The paper demonstrates how to generate naive human pluripotent stem cells from existing induced pluripotent stem cells that may be derived from patients with various diseases, how to generate mouse-human chimeras using these cells and how to quantify the amount of human cells in the chimeras.

“Using our method, one can now track the development of naive human pluripotent stem cells in mouse-human chimeric embryos in real-time,” said Feng. These stem cells can then be manipulated either genetically or pharmacologically, providing valuable information about human development and disease.

“For example, one can label naive human pluripotent stem cells by inserting green fluorescent protein in a hemoglobin gene to study the development of human red blood cells in mouse-human chimeras,” said Feng.

Another application is to generate humanized mouse models to study many human diseases.

“These mice contain critical human cells, tissues or even organs so that they more accurately reflect the human condition,” said Feng. “With our method, the human cells are made along with the mouse during the development of the mouse embryo. There would be better matching and no rejections, because there are ways for the human cells to be made where there is no competition from their mouse counterparts.”

Organs for transplant in the future

By allowing others to improve and adapt the method to eventually generate chimeras in larger animals, this protocol may also lead to the generation of human organs to address the dire shortage of organs available for transplant, said Feng.

“If naive human pluripotent stem cells are able to generate significant amounts of mature human cells in other larger species, it could be possible to make human tissues or even human organs in chimeric animals,” Feng explained.

This would be possible using blastocyst complementation where, Feng explained, normal pluripotent stem cells from one species can reconstitute an organ for that species in a blastocyst of another species that been genetically modified not to grow that particular organ.

Feng added: “Ultimately, a better understanding of how human cells develop and grow in chimeras may enable the generation of human cells, tissues and organs in a completely artificial system and fundamentally change how we treat many human diseases. Research using chimeras is a bridge that must be crossed to reach that possibility.”

Here’s a link to and a citation for the 2021 article,

Generation of mouse–human chimeric embryos by Boyang Zhang, Hanqin Li, Zhixing Hu, Houbo Jiang, Aimee B. Stablewski, Brandon J. Marzullo, Donald A. Yergeau & Jian Feng. Nature Protocols (2021) DOI: https://doi.org/10.1038/s41596-021-00565-7 Published 02 July 2021

This article is behind a paywall.

Here’s a link to and citation for the 2020 work, which led to the publication of the protocols,

Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos by Zhixing Hu, Hanqin Li, Houbo Jiang, Yong Ren, Xinyang Yu, Jingxin Qiu, Aimee B. Stablewski, Boyang Zhang, Michael J. Buck, Jian Feng. Science Advances 13 May 2020: Vol. 6, no. 20, eaaz0298 DOI: 10.1126/sciadv.aaz0298

This paper is open access.

Matrix of gelatin nanofibres for culturing large quantities of human stem cells

A Feb. 14, 2017 news item on ScienceDaily describes work that may have a big influence on stem cell production,

A new nanofiber-on-microfiber matrix could help produce more and better quality stem cells for disease treatment and regenerative therapies.

A matrix made of gelatin nanofibers on a synthetic polymer microfiber mesh may provide a better way to culture large quantities of healthy human stem cells.

Developed by a team of researchers led by Ken-ichiro Kamei of Kyoto University’s Institute for Integrated Cell-Material Sciences (iCeMS), the ‘fiber-on-fiber’ (FF) matrix improves on currently available stem cell culturing techniques.

A Feb. 14/15, 2017 Kyoto University press release (also on EurekAlert), which originated the news item, explains why scientists are trying to find a new way to culture stem cells,

Researchers have been developing 3D culturing systems to allow human pluripotent stem cells (hPSCs) to grow and interact with their surroundings in all three dimensions, as they would inside the human body, rather than in two dimensions, like they do in a petri dish.

Pluripotent stem cells have the ability to differentiate into any type of adult cell and have huge potential for tissue regeneration therapies, treating diseases, and for research purposes.

Most currently reported 3D culturing systems have limitations, and result in low quantities and quality of cultured cells.

Kamei and his colleagues fabricated gelatin nanofibers onto a microfiber sheet made of synthetic, biodegradable polyglycolic acid. Human embryonic stem cells were then seeded onto the matrix in a cell culture medium.

The FF matrix allowed easy exchange of growth factors and supplements from the culture medium to the cells. Also, the stem cells adhered well to the matrix, resulting in robust cell growth: after four days of culture, more than 95% of the cells grew and formed colonies.

The team also scaled up the process by designing a gas-permeable cell culture bag in which multiple cell-loaded, folded FF matrices were placed. The system was designed so that minimal changes were needed to the internal environment, reducing the amount of stress placed on the cells. This newly developed system yielded a larger number of cells compared to conventional 2D and 3D culture methods.

“Our method offers an efficient way to expand hPSCs of high quality within a shorter term,” write the researchers in their study published in the journal Biomaterials. Also, because the use of the FF matrix is not limited to a specific type of culture container, it allows for scaling up production without loss of cell functions. “Additionally, as nanofiber matrices are advantageous for culturing other adherent cells, including hPSC-derived differentiated cells, FF matrix might be applicable to the large-scale production of differentiated functional cells for various applications,” the researchers conclude.

Human stem cells that grew on the ‘fiber-on-fiber’ culturing system

Here’s a link to and a citation for the paper,

Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells by Li Liu, Ken-ichiro Kamei, Momoko Yoshioka, Minako Nakajima, Junjun Li, Nanae Fujimoto, Shiho Terada, Yumie Tokunaga, Yoshie Koyama, Hideki Sato, Kouichi Hasegawa. Biomaterials Volume 124, April 2017, Pages 47–54  http://dx.doi.org/10.1016/j.biomaterials.2017.01.039

This paper is behind a paywall.