Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells

Abstract

Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

Spheroids Formation on Non‐Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches

“…• Solutions preparation is very time consuming • Plate-coating procedure is very time consuming [19,[73][74][75][76][77] www.advancedsciencenews.com www.biotechnology-journal.com dissolve the polymer, at 37 C. [73] Furthermore, Phung et al denoted that the complete evaporation of the ethanol from the poly-HEMA coated plates at RT (15)(16)(17)(18)(19)(20) C) takes 1-2 weeks. [77] To surpass these drawbacks, Lawrenson et al suggested to raise the temperature of the poly-HEMA solution (up to 65 C), in order to facilitate the polymer dissolution and to dry the coating inside a humidity-free incubator at 37 C, for 48 h. Such procedure allows the creation of a uniform, solvent free plate coating.…”

“…In contrast to the agar and agarose solutions, sterile poly-HEMA solutions and poly-HEMA coated plates can be stored at 4 C, up to 2 months, before their used. [73] However, the utilization of poly-HEMA, as a non-adherent material for the production of spheroids through LOT, is a laborious procedure, since it takes several hours (5-7 h) to Figure 2. Overview of the variables studied for the production of spheroids through LOT.…”

“…In this context, the addition of supplements (e.g., viscosityincreasing additives, ECM related components, and others) to the [37,[73][74][75][76] It takes several hours to dissolve poly-HEMA • Put the poly-HEMA solution in a water bath at 65 C [73,77] Poly-HEMA coating drying is slow (1 to 2 weeks at RT)…”

“…[38,61] Poly(2-hydroxyethyl methacrylate) (poly-HEMA) is a nonadherent, soft and flexible plastic that has been recently applied as an alternative polymer to produce spheroids by LOT. [72] Poly-HEMA is usually dissolved in ethanol 95% (100% ethanol does not dissolve the polymer [73] ) and then it is used to coat the materials surfaces. [74][75][76] Moreover, poly-HEMA is sterilized by filtering the sample solution with a 0.22 μm pore membrane filter.…”

Stem cells and clinical practice: new advances and challenges at the time of emerging problems with induced pluripotent stem cell therapies

“…26, 53,56,57 Since it is unlikely that so many PSC and MultiSC types exist in adult tissues, all these early-development stem cells endowed with broader differentiation potential and residing in adult tissues are most likely closely related and represent overlapping populations of cells.…”