Abstract
Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.
Highlights
Human mesenchymal stem cells possess selfrenewal and multilineage differentiation potential [1, 2] and are extensively used in clinical studies. hMSCs can be derived from a wide range of tissues, such as the bone marrow, adipose tissue, umbilical cord, placenta, and dental pulp [3], and can be cultured in vitro for several generations
When cultured in MiPS medium, a medium originally designed for maintenance and expansion of human embryonic stem cells [34, 35], the dissociated single hMSCs maintained a round cell morphology, attached lightly to the tissue culture dish surface at day 1 and generated spheroids at day 3, while the single hMSCs seeded in L-fetal bovine serum (FBS) maintained fibroblast-like morphology (Figure 1(b))
To further determine the key ingredients in MiPS that promote spheroid formation, we conducted a screening that each time, one component was removed from MiPS to establish several incomplete MiPS groups and test the effect of each group on hMSC spheroid formation
Summary
Human mesenchymal stem cells (hMSCs) possess selfrenewal and multilineage differentiation potential [1, 2] and are extensively used in clinical studies. hMSCs can be derived from a wide range of tissues, such as the bone marrow, adipose tissue, umbilical cord, placenta, and dental pulp [3], and can be cultured in vitro for several generations. A variety of in vitro 3D spheroid culture approaches have been developed [11,12,13], including hanging drop [7, 14,15,16], precoating of low-adhesive substrates [17], membranebased aggregation [5, 18, 19], and forced aggregation [20] Most of these methods use conditioned medium containing fetal bovine serum (FBS), which contains undefined components and is not recommended for clinical applications [21, 22]. Several studies using a serum-free medium have successfully generated characterized hMSC
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