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Key Technologies to Realize Broad Use/Practical Use of iPS Cell Technology

iMatrix-511

Manufactured by MATRIXOME, Inc. (Nippi Inc.)

Human recombinant laminin 511 E8 fragment (LM511E8)

iMatrix-511

To facilitate self-renewal and pluripotency of human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in culture, a biologically relevant microenvironment is needed. We at MATRIXOME, Inc. have now produced just such a microenvironment with a combination of extracellular matrix (ECM) and medium components.

The ECM component:

Laminin 511 (LM511) is a natural laminin isoform and is an optimal matrix for hPSC cultivation, since LM511 is ubiquitously expressed throughout the body and binds to α6β1 integrin, a high affinity receptor for LM511 expressed on the hPSCs.

iMatrix-511 is the Matrixome trade name for human recombinant laminin 511 E8 fragment (LM511E8), which is being used successfully for the cultivation of human pluripotent stem cells (hPSCs).

LM511E8 represents a functionally minimal form of LM511 that retains the full binding capacity to α6β1 integrin (Ido et al., 2007). Indeed, additional studies (Miyazaki et al., 2012) found that LM511E8 had significantly higher adhesivity to hPSCs than intact LM511 and other feeder-free matrices, such as vitronectin, fibronectin, and matrigel.

LM511E8’s binding to α6β1 integrin effectively supports hPSC self-renewal and pluripotency in a feeder-free stem cell culture system when used with chemically defined media.

Use of LM511E8 enables stable long-term single cell passaging without karyotypic abnormalities (more than 30 passages to date). Furthermore, LM511E8 facilitates hPSC growth compared to the other matrices mentioned above. For example, hPSCs will expand 100-fold within 6-7 days of cultivation on LM511E8 (Nakagawa et al., 2014).

LM511E8 also supports reprogramming of human primary fibroblasts and blood cells to hiPSCs and differentiation into diverse cell types, including dopaminergic progenitors (Doi et al., 2014), cholangiocytes (Takayama et al., 2016) and retinal pigment epithelial cells (patent numbers: US 20160237403 A1 and US20160244721).

References:

Ido H et al. The requirement of the glutamic acid residue at the third position from the Carboxyl termini of the laminin γ chains in integrin binding by laminins. Journal of Biological Chemisitry, 282(15), 111144-11154. 2007

Miyazaki T et al. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nature communications, 3, 1236. 2012

Doi D et al. Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation. Stem cell reports, 2(3), 337-350. 2014

Takayama K et al. Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells. Biochemical and biophysical research communications, 474(1), 91-96. 2016

For the Future


StemFit®
AK02N・AK03N

StemFit® is a world class induced pluripotent stem cell (iPS)/embryonic stem cell (ES) culture medium that is cost-effective and exhibits optimal performance.
Originally, iPS were cultured with feeder cells, which were often of animal origin.
However, in the clinical setting it is essential that iPS can be cultured using simple methods and that all culture media components are safe.
By combining Ajinomoto’s superior analytical and nutritional techniques together with knowledge about iPS obtained from Japan’s cutting-edge academic institutes, StemFit® was created as a feeder-free medium that allows iPS and ES to grow stably for long periods (it provides the components required for sustaining such cultures at the optimal ratio). There are two StemFit® product lines, StemFit® AK02N, which is designed for use in basic research, and StemFit® AK03N, which is designed for use in clinical research.

StemFit®AK02N

StemFit®AK03N

Background to development

In order to facilitate the rapid development of regenerative medicine, further technological innovations are required together with a cost structure that is compatible with commercial revenues. A large proportion of the cost of stem cell-based regenerative therapies is derived from the media used for cell culturing. Most of the culture media used for regenerative medicine are produced by foreign companies, but these products are not of sufficient quality for use in clinical research into regenerative medicine in Japan. Ajinomoto Co., Inc., successfully developed the StemFit® medium within a short period of time due to the company’s experience of creating a serum-free medium for biopharmaceutical manufacturing; i.e., ASF medium, and clinical nutrients.

Data

1. Cell proliferation curve

Human iPS (strain 201B7) that were grown in StemFit® AK03N exhibited a stable and high proliferation rate for a long period.

Cell proliferation curve

2. Culture tests with various matrices

It has been confirmed that human iPS (strain 201B7) can be grown in StemFit® AK03N on a 6-well plate coated with a commercially available matrix; i.e., iMatrix-511, Vitronectin-N, or Matrigel®. In addition, positivity for the pluripotency markers octamer-binding transcription factor (OCT)3/4 (red) and transcription-associated protein (TRA)-1-60 (green) was detected in the presence of each scaffold.

Culture evaluation with various scaffolds
Culture evaluation with various scaffolds

Technical Details

Technical Details

The prototype of the StemFit® medium was developed by taking advantage of (i) metabolic data about amino acids, (ii) bio-based material manufacturing technology, (iii) compounding agents produced using design technology and high-quality materials, (iv) analytical techniques, and (v) quality control engineering, which were accumulated by Ajinomoto Co., Inc., during the development of clinical nutrients and ASF medium. StemFit® AK03N, which is used in clinical research, is solely composed of purified recombinant protein; i.e., it is free from animal- and human-derived components.

Broad use/practical use

StemFit® AK03N, which is intended for clinical use, has been shown to be cost-effective and to allow the production of long-term, stable cultures with high proliferation rates by the Pharmaceuticals and Medical Devices Agency (PMDA), the pharmaceutical regulatory authority of the Japanese Ministry of Health, Labour and Welfare. It has also been confirmed that this product does not contain any raw materials that are covered by the “Standards for Biological Materials”. By supplying StemFit® for the culturing of iPS and ES, Ajinomoto Co., Inc., is contributing to the realization of health improvements around the world and the development of regenerative medicine and pharmaceutical products.

The future

StemFit® is just one of many factors that are required for the development of regenerative medicine. To facilitate rapid advances in regenerative medicine, it is important that technology platforms and companies that are involved in protein engineering; the production of synthetic materials, cell culture devices, tissue culture devices, containers, or testing equipment; clinical research organizations; and transportation form alliances.
Ajinomoto Co., Inc., is continuing to develop the StemFit® medium, and hence, is contributing to the creation of materials for regenerative medicine by harnessing biomanufacturing technology.


CytoTune®-iPS 2.0, 2.0L

The Sendai virus vector kit that allows for the creation of iPS cells with intact chromosomes and exogenous gene-free

CytoTune®-iPS

CytoTune®-iPS is a product developed by incorporating the so-called Yamanaka’s four genes (OCT3/4, SOX2, KLF4, c-MYC (L-MYC)) required for efficient nucleus initialization into the Sendai virus vector (SeV vector). Through proper use, the artificial pluripotent stem cells (iPS cells) can be induced from human somatic cells. CytoTune®-iPS 2.0 includes c-MYC, whereas CytoTune®-iPS 2.0L includes L-MYC, considered to be safer.CytoTune®-iPS 2.0L kit has the same composition as that of the clinical CytoTune®-iPS.

Background to Development

CytoTune®-iPS 2.0 was developed to enhance the iPS cell induction efficiency and virus RNA elimination speed of CytoTune®-iPS. Moreover, ID Pharma Co., Ltd. initiated the development of the clinical CytoTune®-iPS in response to global customer’s needs.. According to expert opinions, L-Myc was adopted during development instead of the c-Myc used for conventional CytoTune®-iPS in order to reduce the clinical risk and enhance safety. This product is CytoTune®-iPS used for research, which applies the same product configuration as the vector for clinical use..

Technical details

CytoTune®-iPS

[No damage to the chromosome]
The main feature of the Sendai virus is that it has RNA as its genome; however, unlike retrovirus, it stays in the cytoplasm as RNA and the replication, transcription and translation are conducted there. The chromosome will not be damaged because the genetic information does not enter the cell nucleus to be incorporated into the DNA sequence of the host.

[High infectivity]
Another feature of the Sendai virus vector is its high infectivity. The Sendai virus infects by binding the HN protein on the viral surface to sialic acid on the cell membrane. This sialic acid is expressed in most cell types; therefore, the virus can infect many cell types.

[Elimination of the inducing factor]
The inducible genes were seen persistently in the Sendai virus-infected cells due to continuous replication in the cytoplasm; however, the novel technology developed by ID Pharma Co., Ltd. allows it to be eliminated from the cell. In iPS cells induced by CytoTune®-iPS, virus vectors gradually disappeared with passage, resulting in the successful creation of iPS cellswithout virus.

Role in Broad Use/Practical Use

The Sendai virus vector is the most suitable for clinical applications that require a higher level of safety because it is unnecessary to consider the unfavorable transformation such as carcinogenesis when using a retroviral vector. Furthermore, a wide range of animals from mammal to avian species can be targeted due to its high infectivity and. For CytoTune®-iPS 2.0, the induction efficiency was further improved, and we were successful in the induction of iPS cells from refractory cells.

CytoTune®-iPS

CytoTune®-iPS

CytoTune®-iPS 2.0L is the kit for researchers who are considering conducting a study at a stage close to clinical practice while maintaining a high induction efficiency of iPS cells.
As for elimination of the inducing factor, at least 80% of colonies lose vectors within just two passages, similar to CytoTune®-iPS 2.0.

For the Future

iPS cell research is at the stage where it shifts from the research level to clinical practice to establish the differentiation method targeting diseases. This vector progresses to CytoTune®-iPS 2.0L as a non-GMP-compliant product, which is the same product configuration as CytoTune®-iPS 2.0LG that creates clinical iPS cells as a GMP-compliant product. It will be useful to create iPS cells equivalent to the iPS cells used for future clinical practices by oneself to proceed with differentiation research. The technology using the Sendai virus vector has been focused on the induction of iPS cells, but may be utilized for the differentiation induction to various cells/tissues from iPS cells because it is possible to control the ON/OFF expression to some extent.
We are currently proceeding with research and development based on the concept that the features such as [No damage to the chromosome] and [High infectivity] may also play a major role in these fields.