High-cell density-induced VCAM1 expression inhibits the migratory ability of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Download Full-text

Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

Scientific Reports ◽

10.1038/s41598-021-00763-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Minh Nguyen Tuyet Le ◽

Mika Takahi ◽

Kiyoshi Ohnuma

Keyword(s):

Stem Cells ◽

Cell Density ◽

Pluripotent Stem Cells ◽

High Cell Density ◽

High Cell ◽

Cardiomyocyte Differentiation ◽

Paracrine Factors ◽

Induced Pluripotent ◽

Low Cell Density ◽

Cardiac Mesoderm

AbstractCardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell–cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors.

Download Full-text

Adipose tissue-derived mesenchymal stem cells cultured at high cell density express brain-derived neurotrophic factor and exert neuroprotective effects in a 6-hydroxydopamine rat model of Parkinson’s disease

Genes & Genomics ◽

10.1007/s13258-014-0239-0 ◽

2014 ◽

Vol 37 (2) ◽

pp. 213-221 ◽

Cited By ~ 5

Author(s):

Joon Beom Park ◽

Jin Suk Lee ◽

Byung Pil Cho ◽

Ki-Jong Rhee ◽

Soon Koo Baik ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Rat Model ◽

Neurotrophic Factor ◽

High Cell Density ◽

High Cell ◽

Neuroprotective Effects ◽

6 Hydroxydopamine ◽

Cells Cultured

Download Full-text

High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2018.03.007 ◽

2018 ◽

Vol 126 (3) ◽

pp. 379-388 ◽

Cited By ~ 2

Author(s):

Shota Tashiro ◽

Minh Nguyen Tuyet Le ◽

Yuta Kusama ◽

Eri Nakatani ◽

Mika Suga ◽

...

Keyword(s):

Stem Cells ◽

Cell Density ◽

Pluripotent Stem Cells ◽

High Cell Density ◽

Perfusion Culture ◽

Human Pluripotent Stem Cells ◽

Spatial Patterning ◽

High Cell ◽

Culture Chamber ◽

Induced Differentiation

Download Full-text

Integrin-Linked Kinase (ILK) Regulates Urinary Stem Cells Differentiation into Smooth Muscle via NF-κB Signal Pathway

Stem Cells International ◽

10.1155/2021/6633111 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Liang-liang Huang ◽

Jun-hong Deng ◽

Jing-xuan Xie ◽

Zi-bin Lin ◽

Hui Jiang ◽

...

Keyword(s):

Stem Cells ◽

Smooth Muscle ◽

Cell Density ◽

High Cell Density ◽

Specific Marker ◽

High Cell ◽

Limited Life ◽

Key Factor ◽

Unlimited Growth ◽

Integrin Linked Kinase

Objectives. Urinary stem cells (USCs) have the capacity for unlimited growth and are promising tools for the investigations of cell differentiation and urinary regeneration. However, the limited life span significantly restricts their usefulness. This study is aimed at exploring the effect of integrin-linked kinase (ILK) on the smooth muscle cells (SMCs) differentiation of the dog USCs and investigating its molecular mechanism. Methods. An immortalized USCs cell line with the molecular markers and biological functions was prepared. After successfully inducing the differentiation of USCs into SMCs, the expression level of the unique key factor and its mechanisms in this process was determined through real-time polymerase chain reaction, Western blot, or Immunofluorescence staining. Results. We found that high cell density promoted USCs differentiation SMCs, and ILK was necessary for USCs differentiation into SMCs. Knocking down ILK decreased the expression of SMCs specific-marker, while using a selective ILK agonist increased the expression of SMCs specific-marker. Furthermore, ILK regulated SMCs differentiation in part through the activation of NF-κB pathway in USCs. A NF-κB activity assay showed overexpression of ILK could significantly upregulate NF-κB p50 expression, and NF-κB p50 acts as downstream signal molecular of ILK. Conclusion. High cell density induces the differentiation of USCs into SMCs, and ILK is a key regulator of myogenesis. Furthermore, NF-κB signaling pathway might play a crucial role in this process.

Download Full-text