Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

Background Although umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified. Methods Mononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system. Results The results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth. Conclusions UCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Multilineage-differentiating stress-enduring cells alleviate atopic dermatitis-associated behaviors in mice

Background Pruritus is a recurring, long-lasting skin disease with few effective treatments. Many patients have unsatisfactory responses to currently available antipruritic treatments, and effective therapeutics are urgently needed to relieve symptoms. A previous study reported that mesenchymal stem cell (MSC)-mediated immune regulation could be used to treat skin inflammatory diseases. Multilineage-differentiating stress-enduring (Muse) cells are a new type of pluripotent stem cell that may also have the potential to treat inflammatory skin diseases. Methods Muse cells were isolated from human bone marrow-derived MSCs (BMSCs) via the 8-h longterm trypsin incubation (LTT) method. Repeated use of 2,4-dinitrofluorobenzene (DNFB) induced atopic dermatitis (AD) in a mouse model. Immunofluorescence, behavior recording, and image analysis were used to evaluate the therapeutic effect of subcutaneous Muse cell injection. Real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression of inflammatory factors. In vitro, wound healing and cell proliferation experiments were used to examine the effect of Muse cell supernatant on keratinocytes. Results Our results showed that subcutaneous injection of Muse cells after AD model induction significantly alleviated scratching behavior in mice. The evaluation of dermatitis and photos of damaged skin on the back of the neck revealed that Muse cells reduced dermatitis, playing an active role in healing the damaged skin. The activation of spinal glial cells and scratching behavior were also reduced by Muse cell injection. In addition, we also showed that the expression levels of the inflammatory factors interleukin (IL)-6, IL-17α, and IL-33 in both the spinal cord and skin were suppressed by Muse cells. Furthermore, Muse cells not only exerted anti-inflammatory effects on lipopolysaccharide (LPS)-induced human HaCat cells but also promoted wound healing and keratinocyte proliferation. Conclusions In vivo, Muse cells could alleviate scratching symptoms, reduce epidermal inflammation, and promote wound healing. In vitro, Muse cells could also promote the migration and proliferation of keratinocytes. In summary, Muse cells may become a new therapeutic agent for the treatment of AD.

Endothelial regeneration in repair from vascular injury and stem cell biology

Professor Teruo Inoue and his collaborators are exploring repair from vascular and myocardial injury in the context of stem cell biology in work that is set to make waves in regenerative medicine. This research involves endothelial progenitor cells (EPCs) for vascular repair, adipose-derived regenerative cells (ADRCs) for angiogenesis and multilineage differentiating stress enduring cell (Muse) cells for myocardial repair. Inoue and his collaborators are also investigating the 'wound repair priming' phenomenon with a view to overcoming the challenge of the inconsistent capacities of angiogenesis due to individual differences in cell quality. The researchers found that the ADRCs (also called adipose-derived stromal fractions: SVFs) obtained from subcutaneous fat after manipulation caused by surgical injury as well as ischaemia showed higher angiogenetic ability. The researchers plan to pharmacologically reproduce wound repair priming in order to facilitate more consistent cell therapy using ADRCs. The team is also exploring other promising functional analysis methods for stem cells, including comprehensive gene analysis using single cell RNA sequence (scRNA seq), which the researchers plan to apply to Muse cells. In addition to Muse cell research targeting myocardial repair, the team will also conduct Muse cell vascular research as they believe that Muse cell treatment holds great promise for the repair of injured-vessel sites. Ultimately, Inoue and his collaborators hope their work will significantly impact medical research and clinical medicine.

Author Correction: Therapeutic benefit of Muse cells in a mouse model of amyotrophic lateral sclerosis

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Non-Tumorigenic Pluripotent Reparative Muse Cells Provide a New Therapeutic Approach for Neurologic Diseases

Muse cells are non-tumorigenic endogenous reparative pluripotent cells with high therapeutic potential. They are identified as cells positive for the pluripotent surface marker SSEA-3 in the bone marrow, peripheral blood, and connective tissue. Muse cells also express other pluripotent stem cell markers, are able to differentiate into cells representative of all three germ layers, self-renew from a single cell, and are stress tolerant. They express receptors for sphingosine-1-phosphate (S1P), which is actively produced by damaged cells, allowing circulating cells to selectively home to damaged tissue. Muse cells spontaneously differentiate on-site into multiple tissue-constituent cells with few errors and replace damaged/apoptotic cells with functional cells, thereby contributing to tissue repair. Intravenous injection of exogenous Muse cells to increase the number of circulating Muse cells enhances their reparative activity. Muse cells also have a specific immunomodulatory system, represented by HLA-G expression, allowing them to be directly administered without HLA-matching or immunosuppressant treatment. Owing to these unique characteristics, clinical trials using intravenously administered donor-Muse cells have been conducted for myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, perinatal hypoxic ischemic encephalopathy, and amyotrophic lateral sclerosis. Muse cells have the potential to break through the limitations of current cell therapies for neurologic diseases, including amyotrophic lateral sclerosis. Muse cells provide a new therapeutic strategy that requires no HLA-matching or immunosuppressant treatment for administering donor-derived cells, no gene introduction or differentiation induction for cell preparation, and no surgery for delivering the cells to patients.

MUSE Stem Cells Can Be Isolated from Stromal Compartment of Mouse Bone Marrow, Adipose Tissue, and Ear Connective Tissue: A Comparative Study of Their In Vitro Properties

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.