Induced pluripotent stem cells (iPS cells) are regarded as the "universal cells" of regenerative medicine because of their functions such as infinite proliferation and multi-directional differentiation potential. In Japan, the induced pluripotent stem cell (iPSC) storage project began in 2013. The goal of the project is to produce and release a clinic-grade HLA homozygous iPSC cell line that can cover almost the entire Japanese population.

1. Introduction

In 2012, Japan listed key areas such as regenerative medicine as priority areas in accordance with the national strategy of the "Comprehensive Strategy for Japan's Rejuvenation" and committed to providing long-term support for advanced research in the field of induced pluripotent stem cells (iPSCs).

The 2012 Nobel Prize in Physiology or Medicine was awarded to Nobuya Yamanaka "for discovering that mature cells can be reprogrammed into pluripotent cells", further accelerating the country's support for the policy.

The Japanese government subsequently launched a new initiative to "realize a network of research centers for regenerative medicine" that will last for ten years starting in Fiscal Year 2013 (FY). The program consists of the iPSC Stock Project, which aims to establish an iPSC haplobank homozygous for human leukocyte antigen (HLA) and support related research and development projects.

Kyoto University's iPS Cell Research and Applications Center (CiRA) was selected as the core center of the iPSC Stock Project, including a large number of companies and medical institutions participating. The iPSC Stock Project provides high-quality clinical grade cell lines as an infrastructure for innovative research. In addition, for any cell transplant, HLA matching is required to reduce immune rejection. Therefore, the goal of the iPSC Stock Project is to manufacture and release HLA homozygous iPSC cells that can cover almost the entire Japanese population by the end of fiscal year 2022.

2. Summary of cell line distribution and conditions

The iPSC Stock Project uses two types of donor cell sources: adult peripheral blood mononuclear cells (PBMC) and cord blood cells (CBC). All PBMC and CBC are collected after obtaining written consent from the donor. Donor screening and eligibility determination are conducted in accordance with Japanese regulations. Inclusion criteria included PBMCs 20 years of age or older and homozygosity for three HLA gene loci: HLA-A, -B, and-DR. Positive testing for HBV, HCV, HIV HTLV-1, parvovirus B19, Treponema pallidum, or cytomegalovirus infection is an exclusion criterion. Subjects with diseases such as bacterial or protozoal infections, sepsis, malignancy, infectious spongiform encephalopathy, or overt genetic diseases were excluded through medical interviews and examinations.

CiRA owns a cell processing facility called FiT (iPS Cell Therapy Facility) and a cell product manufacturing facility license issued by the Japanese government for clinical research. CiRA has developed a feeder and xenogene-free culture system at FiT to comply with Japanese regulatory requirements.

Using this system, iPSC was generated by electroporation of episomal plasmid vectors into which reprogrammed genes (pCE-OCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD and PCXB-EBNA1) were introduced. iPSC has been expanded to produce primary and secondary cell stocks (PCS and SCS), which are cryopreserved for long-term storage. iPSC inventory includes research-grade and clinical grade cells, and iPSC SCS is provided as clinical grade inventory. As of April 2019, the project's HLA types cover approximately 40% of the Japanese population.

Based on donor requirements for iPSC storage, a third-party committee reviews the purpose of the study, ethical conditions, personal information protection policies, past achievements and facilities based on donor consent. If approved, iPSC storage will be provided accordingly. For non-profit organizations, iPSC storage is free. For for-profit organizations, storage costs for research-grade and clinical grade iPSC are 50,000 yen (approximately US$500) and 100,000 yen (US$1,000), respectively, plus additional costs.

3. Quality Control Test Summary

Prior to the release of iPSC for storage, negative testing for microorganisms (sterile), mycoplasma, endotoxin, and human viruses (including HBV, HCV, HIV HTLV, and parvovirus B19), as well as morphology, HLA genotyping, and STR analysis were confirmed. In addition, reference tests were performed to obtain good clone selection. These tests include proliferation ability, post-thaw viability and cell count (NucleoCounter NC-200), pluripotency markers, karyotype analysis, single nucleotide mutation/insertion deletion mutation (SNV/Indel) and (copy number variation (CNV) analysis, which are consistent with the Cancer-Related Database (Catalog of Somatic Mutation in Cancer (COSMIC) Cancer Gene Census (Sondka et al., 2018) and Shibata List (http://www.pmda.go.jp/files/000152599.pdf) and clone structure analysis. When iPSC inventory is shipped, a certificate of analysis is provided that contains donor information and test results details for each iPSC inventory cell line.

4. future plans

Using a stock of HLA-homozygous iPSC, CiRA generated new HLA-C-preserved iPSC to inhibit natural killer cell responses and maintain HLA expression and antigen presentation through genome editing techniques.

CiRA's first iPS cell stocks were held in 2015, and the first clinical trial involving iPS cells began in 2017. Since then, more than 10 clinical trials using the CiRA Foundation's iPS cell reserve have begun and positive results have been reported. The CiRA Foundation plans to continue sharing its resources with organizations around the world to develop new medical approaches. Because of the importance of HLA in donor-patient matching, the CiRA Foundation is exploring HLA genome editing so that more patients in Japan and abroad can qualify for iPS cell-based therapies. In addition, further research is underway on the production and quality of iPS cells made from patient cells. This is part of an iPS project that works with multiple companies and universities. In addition, CiRA is developing a low-cost native iPSC storage project, which CiRA calls a personal iPS project, which provides preparation storage services to individuals and families. CiRA will simultaneously handle clinical-grade iPSC inventory, genome editing inventory and individual iPS projects.