1. Current Status and Issues
   Mesenchymal stem cells (MSCs) are tissue stem cells that are expected to be the next most clinically applicable after hematopoietic stem cells because they have fewer ethical issues associated with cell collection and possess diverse differentiation potential, including bone, cartilage, and fat. Because they can be isolated using relatively simple procedures, they are widely used as biomaterials, primarily for in vitro differentiation into cartilage and bone followed by local transplantation. Investigator-initiated clinical trials have begun at several institutions. In the United States, more than 300 clinical trials are currently underway (Yano Economics Report, 2004). Currently available mesenchymal stem cells are obtained by seeding bone marrow mononuclear cells onto culture dishes and recovering the fibroblast-like colony-forming cells (CFU-F) that emerge after 2–3 weeks of culture (see figure below).
   However, there are several issues with the adherent cell population obtained using this method.
   1. Differentiation potential: Contamination with blood cells and contaminant cells with low or no differentiation potential is unavoidable, and undifferentiated cells with the ability to differentiate and proliferate (high-quality MSCs) are actually contained at a low frequency of approximately 1-3/1x103 (Colter PNAS 2000).
   2. Proliferation potential: MSCs, which are primary cultured cells, have a limited proliferation rate and usually lose their proliferation ability after 3-4 passages. At that time, their differentiation potential also decreases significantly.
   3. Migratory ability: Cell phenotypes change during culture expansion, and MSCs that maintain migratory ability within the bone marrow lose this ability during in vitro culture (Rombouts Leukemia 2003, Morikawa J.Exp.Med. 2009). For this reason, MSCs cannot be administered intravenously to treat systemic diseases.

   Therefore, for safe and effective cell therapy, it is necessary to ensure the inherent cell functions of MSCs, namely, not only the proliferation and differentiation abilities that have traditionally been used as indicators, but also the uniformity and migration ability of the cells.

2. Identification and Isolation of High-Quality MSCs in Human Bone Marrow
   Principal Investigator Matsuzaki et al. demonstrated that the use of two antibodies, LNGFR (CD271) and Thy1 (CD90), enables highly efficient selection of human MSCs through the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Regenerative Medicine Realization Project. They then developed a technology for directly isolating human MSCs from bone marrow, peripheral blood, placental chorion, and dental pulp using a cell sorter (Mabuchi et al. Stem Cell Reports 2013). LNGFR Thy-1 co-positive cells (LT cells) form fibroblast-like colonies at an extremely high frequency of 1 in 6. Compared to whole bone marrow mononuclear cells, the colony-forming cells are enriched approximately 30,000-fold, making this the isolation method with the highest enrichment rate in the world. After these LT cells were separated into single cells in a 96-well plate (B), the proliferation rate of each well was compared, and the cells were classified into rapidly expanding clones ( REC ) that reached confluence after 2 weeks and other clones ( Medium / Slow: MEC /SEC) (C).(Rapidly Expanding Clone: REC)(Medium/Slow: MEC/SEC)(C)。
   
   A: MSCs in human bone marrow exist in the LNGFR Thy1 co-positive fraction (LT cells).
   B: Single-cell isolation of LT cells.
   C: MSC clones obtained by monoculture of LT cells can be classified into three groups based on differences in proliferation rate.

   

   Compared to MECs /SECs,REC/ are high-quality, high-purity human MSCs that overcome all of the problems previously posed by them, including: A) they are a homogeneous cell population; B) they do not show cellular senescence; C) the majority of the cells are in the proliferation phase; D) they exhibit high differentiation potential (especially adipogenic differentiation potential); and E) they exhibit migratory properties (Mabuchi et al. Stem Cell Reports 2013).
   

   Characteristics of REC cell performance
   A: REC cell morphology is small and uniform. B: They are fresh cells that do not stain with SA-β-gal. C: The majority of RECs are positive for Ki67, a cell marker in the proliferation phase. D: Both osteogenic and adipogenic differentiation are good. E: After intravenous administration to experimental animals, no cells were observed trapped in the pulmonary capillaries.
3. Overview of Isolation of Highly Purified Human MSCs (RECs)
   Matsuzaki et al. have demonstrated that similar cell populations can be isolated not only from bone marrow but also from adipose tissue and placental chorion. Based on these results, they established a procedure for obtaining highly purified MSCs（=REC）
   1. Preparation of mononuclear cells from bone marrow (or fat/placental chorion)
   2. After staining with anti-LNGFR and Thy-1 antibodies, LT cells were single-cell sorted into 96-well plates.
   3. After 2 weeks, wells that reached confluence were selected (RECselection).
   4. Cell collection and cryopreservation

   

   Following this protocol allows for consistent production of highly purified MSCs (Patent Application 2013-161752 / Method for Isolation and Cultivation of Human Mesenchymal Stem Cells / Matsuzaki Yumi et al. 3). Furthermore, by seeding cells recovered from one well onto a 35mm culture dish and culturing for 7-10 days, a homogenous cell population with high differentiation and proliferation potential of approximately 5x106 cells is obtained. Even after a certain period of culturing and expansion, differentiation and migration capabilities are maintained. Furthermore, the cells can be stored as frozen cells, and have already been provided to numerous research facilities as research cells. REC has an extremely high cell performance and virtually no variation between lots, resulting in an extremely favorable user reputation. Further growth is expected with its official launch on the market.

As mentioned in (1), MSCs (also known as CFU-Fs) isolated using conventional methods contain not only RECs, which have extremely high cellular activity, but also MECs/SECs, which have undergone some degree of cellular senescence (see lower figure).

While RECs should be the only target for both research and clinical use, isolating RECs requires the single-cell isolation and clonal culture techniques established by the principal investigators. Single-cell isolation using a cell sorter is a highly specialized technique and has not yet become widely adopted. Therefore, we recognized the urgent need to develop markers that specifically recognize RECs and their corresponding antibodies, and conducted the following research and development.

Medical and Biological Laboratories Co., Ltd. (formerly ACTGen Co., Ltd.; merged with ACTGen in March 2013) possesses a patented technology (signal sequence trap method, see lower figure) that comprehensively identifies membrane protein and secretory protein genes from any cell sample and develops monoclonal antibodies against them. This technology not only allows us to obtain genetic information on cell surface markers, but also enables the continuous development of candidate monoclonal antibodies for cell separation, which is our ultimate goal. In an industry-academia collaboration project (JST manifestation stage) that aims to further develop the technological seeds of the principal investigators into results of great significance for industrial application, we identified RECs-specific membrane proteins and produced monoclonal antibodies for five of them (lower figure).

In additional studies after the manifestation stage, functional analysis of Fzd5 and its co-receptor Ror2 was completed, confirming that they are proteins essential for the undifferentiated state of RECs and that their expression is REC-specific (lower figure).

1. Human bone marrow-derived MSCs obtained by conventional methods As mentioned above, currently commercially available human MSCs are cell populations obtained by conventional adherent culture, and most of the cells contained in frozen vials have low differentiation potential, divide slowly, and can only divide a limited number of times. Furthermore, it is well known that output varies depending on culture conditions and individual techniques, making it difficult to ensure reproducibility for clinical application.
2. Human adipose tissue-derived MSCs
   have recently attracted attention as cells that exhibit properties similar to human bone marrow-derived MSCs, and frozen vials similar to those of bone marrow-derived MSCs are beginning to be distributed. However, like the bone marrow-derived MSCs mentioned above, the isolation method used for these cells results in a cell population that is culture-isolated and contains many impurities. Furthermore, while these cells have superior adipocyte differentiation potential compared to bone marrow-derived MSCs, they have lower bone and cartilage differentiation potential, which is expected to limit their applications.
3. Muse cells
   Muse cells are cells obtained by culturing bone marrow mononuclear cells into cell masses called M-clusters, and are said to have the ability to differentiate across germ layers, similar to ES/iPS cells (Kuroda, PNAS, 2010). Although their differentiation potential surpasses that of human bone marrow-derived MSCs, M-clusters have extremely low proliferation rate and do not proliferate at all, making it expected to be difficult to secure sufficient numbers of cells for clinical use.
   
   Characteristics of REC cell performance (proliferation)
   A: RECs are a cell population with extremely high proliferation potential, and one REC can be cultured and expanded to 1x1012 cells. Compared to MSCs obtained by conventional methods from the same number of bone marrow mononuclear cells, 1x106 times the cell number can be obtained. B: The proliferation potential of Muse cells contained in M-clusters is extremely low, and proliferation stops after two weeks, meaning that the maximum number of Muse cells that can be obtained from one cell is 40 (from PNAS 2010).
4. Pluripotent stem cell (ESC/iPSC)-derived MSCs
   Several reports have been published on MSCs derived from pluripotent stem cells (ES cells/iPS cells) (Giuliani Blood 2011, Liu ProsOne 2013). However, as with isolation from bone marrow mononuclear cells, a homogenous cell population with excellent differentiation, proliferation, and migration properties has not yet been obtained. Furthermore, the number of days required for induction, isolation, and culture is longer than that required for isolation from bone marrow mononuclear cells. Furthermore, the risk of contamination with undifferentiated cells and the resulting risk of teratoma formation has not yet been eliminated. While promising, this approach is currently not practical.

1. Commercialization of frozen vials of high-purity mesenchymal stem cells and establishment of a contract service.
   High-purity mesenchymal stem cells purified using the cell separation technology originally developed by Matsuzaki et al. will be sealed in frozen vials and commercialized as mesenchymal stem cell products for research use. Furthermore, with a view to adapting them to transplantation medicine, we will establish a system to ensure stable supply of high-purity stem cells with guaranteed cell performance and GMP-compliant levels, build a clinical-grade high-purity MSC separation system, and provide contract cell separation and culture services to clinical facilities.
2. Mesenchymal stem cell isolation and evaluation reagents, cell culture reagents
   Among the monoclonal antibodies specifically staining high-purity mesenchymal stem cells newly developed during the manifestation stage, candidates suitable for cell separation are bound to nanomagnetic particles and commercialized as mesenchymal stem cell isolation reagents. Furthermore, cell evaluation reagents are required to verify the quality of isolated mesenchymal stem cells. We commercialize fluorescent-conjugated antibodies and cell staining reagents as reagents for evaluating the cell quality of mesenchymal stem cells for transplantation. Furthermore, because mesenchymal stem cells require a stable culture environment for their self-proliferation and differentiation capabilities when applied to medical applications, we also offer optimized cell culture reagents.
   

1. High-Purity Mesenchymal Stem Cells
   As mentioned above, mesenchymal stem cell transplantation has already been applied clinically. However, the cells isolated using existing methods only contain 0.1-1% stem cells, leaving purity an issue. According to the "Domestic Mesenchymal Stem Cell-Related Regenerative Medicine Market Forecast (Yano Research Institute, Ltd.: 2004)," the regenerative medicine market is expected to reach 112,000 cases in 2010 and 218,720 cases in 2015. As developed countries continue to age, the number of patients is expected to continue to increase. Furthermore, the regenerative medicine market in the United States is estimated to grow to 600 billion yen by 2020, and the social and economic impact of regenerative medicine using highly purified mesenchymal stem cells is expected to be significant. The figure on the right presents some data comparing image025four currently commercially available products (frozen human bone marrow-derived MSCs) with RECs . When 5 x 106 cells were cultured for four days, RECs achieved average results, ranking third out of five in terms of cell proliferation rate. However, the top-ranked cells, CA and SC, lacked the ability to differentiate into fat cells and lacked full multi-differentiation potential. Furthermore, PC cells, which had a proliferation rate comparable to RECs cells, only possessed the ability to differentiate into bone. As such, human bone marrow-derived MSCs on the market vary in proliferation and differentiation ability, resulting in significant quality differences between lots and products, which can affect experimental accuracy and reproducibility when used in research. Clinically, they lack consistency in terms of therapeutic efficacy. We believe that bringing RECs to market will offer significant advantages, as it overcomes all of the challenges posed by currently widely used MSC cells.
2. ClinicalRECContract Service
   The enactment of the Act on the Safety of Regenerative Medicine, etc., which allows medical institutions to outsource the culturing and processing of cells for regenerative medicine, has made it easier to develop contract services by establishing facilities and procedures that comply with safety standards for cell culturing and processing. Clinical facilities, such as university hospitals, isolate RECs from bone marrow (or placental chorion/adipose tissue) collected from patients , expand them under GMP-compliant culture conditions, and use them in clinical research for transplantation therapy. Research results are shared with the clinical facilities receiving the supplies, and know-how on cell quality control, quality evaluation, and quality standard setting is accumulated, which will be utilized in future clinical development toward pharmaceutical approval as a regenerative medicine product. AIST, which has already achieved clinical MSC isolation, has completed technology transfer of cell culture procedures and other know-how within the CPC to the Shimane University Hospital Transfusion Department, establishing GMP-level culture operations and procedures for conventional MSCs using an isolator installed within the university hospital. Through this project, the goal is to establish a closed-system RECs isolation procedure and operate the facility as a contract facility.
3. Antibodies for Mesenchymal Stem Cell Quality Assessment
   Unlike conventional small molecule drugs, cell therapy, which uses cells derived from tissue donors that vary from batch to batch, is recognized as challenging to standardize. The inability to identify the main component by physicochemical means, such as a specific molecular weight or structural formula, is the most significant difference between cells and small molecule drugs when considering formulation standardization. Because the main component of cell therapy is the cells themselves, standardization is crucial due to factors such as donor origin and the influence of the manufacturing process. Therefore, the development of markers that can be used to produce mesenchymal stem cells of a uniform grade is urgently needed, especially regarding gene expression, which directly correlates with clinical efficacy. CD markers are used as one definition of mesenchymal stem cells. By incorporating functional markers essential for cell function into manufacturing and release testing standards, combined with CD markers, standardization of cell functionality becomes possible. To this end, we believe that the monoclonal antibodies we have developed against a group of genes involved in maintaining the undifferentiated state of MSCs are highly promising.