The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4 , Sox2 , Klf4 , and c-Myc , iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

Rationale:: Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3 ⁺ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction. Objective:: The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair. Methods and Results:: In vivo dynamics of Nano-lantern–labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]–specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]–introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)–S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4 -gene–silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. Conclusions:: Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.

Background and Purpose—: Multilineage-differentiating stress-enduring (muse) cells are endogenous nontumorigenic stem cells with pluripotency harvestable as pluripotent marker SSEA-3 ⁺ cells from the bone marrow from cultured bone marrow-mesenchymal stem cells. After transplantation into neurological disease models, muse cells exert repair effects, but the exact mechanism remains inconclusive. Methods—: We conducted mechanism-based experiments by transplanting serum/xeno-free cultured-human bone marrow-muse cells into the perilesion brain at 2 weeks after lacunar infarction in immunodeficient mice. Results—: Approximately 28% of initially transplanted muse cells remained in the host brain at 8 weeks, spontaneously differentiated into cells expressing NeuN (≈62%), MAP2 (≈30%), and GST-pi (≈12%). Dextran tracing revealed connections between host neurons and muse cells at the lesioned motor cortex and the anterior horn. Muse cells extended neurites through the ipsilateral pyramidal tract, crossed to contralateral side, and reached to the pyramidal tract in the dorsal funiculus of spinal cord. Muse-transplanted stroke mice displayed significant recovery in cylinder tests, which was reverted by the human-selective diphtheria toxin. At 10 months post-transplantation, human-specific Alu sequence was detected only in the brain but not in other organs, with no evidence of tumor formation. Conclusions—: Transplantation at the delayed subacute phase showed muse cells differentiated into neural cells, facilitated neural reconstruction, improved functions, and displayed solid safety outcomes over prolonged graft maturation period, indicating their therapeutic potential for lacunar stroke.

Objective: Muse cells reside as pre-existing pluripotent-like stem cells within the fibroblasts, are nontumorigenic, exhibit differentiation capacity into triploblastic-lineage cells, and replenish lost cells when transplanted in injury models. Cell fate and function of human skin fibroblast-derived Muse cells were evaluated in a rat stroke model. Methods: Muse cells (30,000), collected by pluripotent surface marker stage-specific embryonic antigen-3, were injected stereotaxically into three deposits within the rat ischemic cortex at 2 days after transient middle cerebral artery occlusion, and the cells' biological effects were examined for more than 84 days. Results: Muse cells spontaneously and promptly committed to neural/neuronal-lineage cells when cocultured with stroke brain slices. Muse-transplanted stroke rats exhibited significant improvements in neurological and motor functions compared to control groups at chronic days 70 and 84, without a reduction in the infarct size. Muse cells survived in the host brain for up to 84 days and differentiated into NeuN (∼65%), MAP-2 (∼32%), calbindin (∼28%), and GST-π (∼25%)-positive cells in the cortex, but glial fibrillary acidic protein-positive cells were rare. Tumor formation was not observed. Muse cells integrated into the sensory-motor cortex, extended their neurites into cervical spinal cord, and displayed normalized hind limb somatosensory evoked potentials. Interpretation: Muse cells are unique from other stem cells in that they differentiate with high ratio into neuronal cells after integration with host brain microenvironment, possibly reconstructing the neuronal circuit to mitigate stroke symptoms. Human fibroblast-derived Muse cells pose as a novel source of transplantable stem cells, circumventing the need for gene manipulations, especially when contemplating autologous cell therapy for stroke.

While mesenchymal stem cells (MSCs) are easily accessible from mesenchymal tissues, such as bone marrow and adipose tissue, they are heterogeneous, and their entire composition is not fully identified. MSCs are not only able to differentiate into osteocytes, chondrocytes, and adipocytes, which belong to the same mesodermal lineage, but they are also able to cross boundaries between mesodermal, ectodermal, and endodermal lineages, and differentiate into neuronal- and hepatocyte-like cells. However, the ratio of such differentiation is not very high, suggesting that only a subpopulation of the MSCs participates in this cross-lineage differentiation phenomenon. We have identified unique cells that we named multilineage-differentiating stress-enduring (Muse) cells that may explain the pluripotent-like properties of MSCs. Muse cells comprise a small percentage of MSCs, are able to generate cells representative of all three germ layers from a single cell, and are nontumorigenic and self-renewable. Importantly, cells other than Muse cells in MSCs do not have these pluripotent-like properties. Muse cells are particularly unique compared with other stem cells in that they efficiently migrate and integrate into damaged tissue when supplied into the bloodstream, and spontaneously differentiate into cells compatible with the homing tissue. Such a repairing action of Muse cells via intravenous injection is recognized in various tissues including the brain, liver, and skin. Therefore, unlike ESCs/iPSCs, Muse cells render induction into the target cell type prior to transplantation unnecessary. They can repair tissues in two simple steps: collection from mesenchymal tissues, such as the bone marrow, and intravenous injection. The impressive regenerative performance of these cells provides a simple, feasible strategy for treating a variety of diseases. This review details the unique characteristics of Muse cells and describes their future application for regenerative medicine.

Muse cells, a novel type of nontumorigenic pluripotent-like stem cells, reside in the bone marrow, skin, and adipose tissue and are collectable as cells positive for pluripotent surface marker SSEA-3. They are able to differentiate into cells representative of all three germ layers. The capacity of intravenously injected human bone marrow-derived Muse cells to repair an immunodeficient mouse model of liver fibrosis was evaluated in this study. The cells exhibited the ability to spontaneously differentiate into hepatoblast/hepatocyte lineage cells in vitro. They demonstrated a high migration capacity toward the serum and liver section of carbon tetrachloride-treated mice in vitro. In vivo, they specifically accumulated in the liver, but not in other organs except, to a lesser extent, in the lungs at 2 weeks after intravenous injection in the liver fibrosis model. After homing, Muse cells spontaneously differentiated in vivo into HepPar-1 (71.1±15.2%), human albumin (54.3±8.2%), and anti-trypsin (47.9±4.6%)-positive cells without fusing with host hepatocytes, and expressed mature functional markers such as human CYP1A2 and human Glc-6-Pase at 8 weeks after injection. Recovery in serum, total bilirubin, and albumin and significant attenuation of fibrosis were recognized with statistical differences between the Muse cell-transplanted group and the control groups, which received the vehicle or the same number of a non-Muse cell population of MSCs (MSCs in which Muse cells were eliminated). Thus, unlike ESCs and iPSCs, Muse cells are unique in their efficient migration and integration into the damaged liver after intravenous injection, nontumorigenicity, and spontaneous differentiation into hepatocytes, rendering induction into hepatocytes prior to transplantation unnecessary. They may repair liver fibrosis by two simple steps: expansion after collection from the bone marrow and intravenous injection. A therapeutic strategy such as this is feasible and may provide significant advancements toward liver regeneration in patients with liver disease.