Donor Muse Cell Treatment Without HLA-Matching Tests and Immunosuppressant Treatment

Abstract

The strength of stem cell therapy is the regeneration of tissues by synergistic pleiotropic effects. Among many stem cell types, mesenchymal stem cells (MSCs) that are comprised of heterogenous population are widely used for clinical applications with the expectation of pleiotropic bystander effects. Muse cells are pluripotent-like/macrophage-like stem cells distributed in the bone marrow, peripheral blood, and organ connective tissues as cells positive for the pluripotent surface marker stage-specific-embryonic antigen -3. Muse cells comprise ~1% to several percent of MSCs. While Muse cells and MSCs share several characteristics, such as mesenchymal surface marker expression and their bystander effects, Muse cells exhibit unique characteristics not observed in MSCs. These unique characteristics of Muse cells include selective homing to damaged tissue after intravenous injection rather than being trapped in the lung like MSCs, replacement of a wide range of damaged/apoptotic cells by differentiation through phagocytosis, and long-lasting immunotolerance for donor cell use. In this review, we focus on the basic properties of Muse cells clarified through preclinical studies and clinical trials conducted by intravenous injection of donor-Muse cells without HLA-matching tests or immunosuppressant treatment. MSCs are considered to differentiate into osteogenic, chondrogenic, and adipogenic cells, whereas the range of their differentiation has long been debated. Muse cells may provide clues to the wide-ranging differentiation potential of MSCs that are observed with low frequency. Furthermore, the utilization of Muse cells may provide a novel strategy for clinical treatment.

Keywords: amyotrophic lateral sclerosis; epidermolysis bullosa; immunotolerance; intravenous injection; myocardial infarction; pluripotent; sphingosine-1-phosphate; stroke.

Graphical Abstract

Figure 1.

Muse cell characteristics. (A) Muse cells selectively migrate to the site of damage following intravenous administration. A rabbit AMI model 3 days after intravenous injection of nano-lantern-Muse and non-Muse cells exhibited specific homing of Muse cells to the post-infarct heart tissue, while non-Muse cells were trapped in the lung and rarely homed to the heart. (B) Differentiation of Muse cells into triploblastic-lineage cells by phagocytosis. (C) The mechanism underlying how Muse cells recycle signals from the up-taken damaged/apoptotic cells necessary for differentiation, such as transcription factors. (D) Single-cell RNA sequencing of human Muse cells (original Muse cells) after phagocytosing apoptotic cell fragments of mouse hepatic- (hepatic), mouse cardiac- (cardiac), and rat neural- (neuronal) cells (7 days). Gene expression in differentiated Muse cells is distinct and differs from that in original Muse cells. (E) Cardiac marker expression in quantitative PCR in human Muse cells after phagocytosing apoptotic mouse cardiac cell fragments. (F) Functional assessment of Muse cell-derived cardiac cells after phagocytosis. Intracellular calcium dynamics in green fluorescent protein (GFP)-based Ca calmodulin probe (GCaMP)-h-Muse cells after biochemical depolarization with 70 mM KCl. (G) Evaluation of fibrosis by Sirius red staining in a mouse liver chronic fibrosis model with intravenous injection of human Muse cells, -non-Muse cells, or vehicle at 8 weeks. Anti-fibrosis effect was prominent in Muse cells. Figures were redrawn from Yamada et al, Wakao et al, and Iseki et al with permission.

Figure 2.

Strategy for Muse cell treatment. Muse cells have several outstanding characteristics beneficial for clinical application; (1) HLA-mismatched donor Muse cells can be directly administered to patients without immunosuppressant, due to specific immunotolerance, (2) because Muse cells selectively migrate to damage sites by S1P-S1P receptor 2 axis, intravenous drip is a more efficient method of delivering them to the damaged site than a surgical approach, (3) unlike embryonic stem and induced pluripotent stem cells, Muse cells do not require differentiation induction because they can differentiate into the same cell type as the damaged/apoptotic cell by phagocytosis to replace damaged/apoptotic cells with healthy functioning cells, (4) Muse cells remain in the homed tissue for an extended period without rejection, and thus their bystander effects such as anti-apoptotic and anti-fibrosis effects as well as neovascularization are long lasting.

Figure 3.

Preclinical and clinical trials in AMI and EB. (A-B) AMI rabbit model that received intravenous injection of Muse cells. (A) Nano-lantern–labeled Muse cell homing was substantially inhibited by co-injection of S1P receptor 2-specific antagonist JTE-013. (B) Masson trichrome staining of LV in the vehicle, autologous-Muse cells, -non-Muse cells, and -MSC groups at 2 months. Substantial reduction of infarct size and improvement in cardiac function (LVEF) was recognized at 2 months. Figures were redrawn from Yamada et al with permission. *P < .05, **P < .01, ***P < .001. (C) The first-in-human clinical trial of donor Muse cells in AMI. Changes in LVEF and wall motion score index before and after (until 12 weeks) the administration of CL2020. *P < .05, ***P < .001. Figures were redrawn from Noda et al with permission. (D) Col17-KO EB model mouse that received human Muse cells and CL2020 by intravenous injection. Col17-KO mouse received a single dose of CL2020 and skin samples were collected 4 weeks later. A linear deposition of hCOL7 and hCOL17 in the BMZ (arrowheads) was recognized. Bars. 100 mm. Figures were redrawn from Fujita et al with permission. (E-F) Clinical trial in EB representative clinical images of the patient (patient 1, right lower leg). The area of the erosion rapidly improved after CL2020 administration. Change in the combined size of selected ulcers from the baseline (cm²) per patient. *P < .05. Figures were redrawn from Fujita et al with permission.

Figure 4.

Preclinical and clinical trials in stroke and ALS. (A-B) Immunodeficient mouse with lacunar infarction received human Muse cell injection at 2 weeks. Differentiation of GFP-Muse cells into neuronal (NeuN, MAP2)-, oligodendrocyte (GST-pi)-, astroglial (GFAP)-, microglial (Iba-1)-, and proliferative cell (ki-67)-markers. (C) Loss of function experiment by intraperitoneal injection of diphtheria toxin (DT). Figures A-C were redrawn from Uchida et al, 2017 with permission. (D-F) Clinical trial in subacute stroke. (D) Responder analysis of patients with modified Rankin Scale (mRS) scores of 0-2 at 12 weeks, demonstrating mean (95% CI) response rates in the CL2020 (40.0% [21.1, 61.3]) and placebo (10.0% [0.3, 44.5]) groups; the threshold response rate of 8.7% based on registry data. The lower limit of the 95% CI in the CL2020 group was higher than the 8.7% seen in the registry data. The difference between the CL2020 and placebo groups was analyzed by Fisher’s exact test (mid-P value). (E) Adjusted mean (95% CI) change from baseline in Fugl–Meyer Motor Scale (FMMS) upper limb score, and (F) lower limb score in the CL2020 (n = 19-25) and placebo (n = 8–10) groups. **P < .01, ***P < .001. Figures were redrawn from Niizuma et al with permission. (G) Mouse ALS model that received intravenous injection of human Muse cells. Distribution of nano-lantern-human MSCs and Muse cells in the cervical and lumbar spinal cord 7 days after intravenous injection. Figures were redrawn from Yamashita et al with permission. (H) Clinical trial in ALS. Longitudinal follow-up of the changes in the ALSFRS-R scores. Mean (SD) ALSFRS-R scores of patients with ALS treated with CL2020. Arrowheads indicate the intravenous administration of CL2020. ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; mo, months. Figures were redrawn from Yamashita et al with permission.