Cell cycle regulation by ankyrin repeat-rich membrane spanning scaffold protein and size-dependent fractionation of human mesenchymal stem cells using microfluidic chip filtration

Abstract

As one of the adaptor proteins, ankyrin rich membrane spanning protein (ARMS/Kidins220) is highly expressed in the nervous system, such as the hippocampus, olfactory burb, and motor neurons in the spiral cord. Generally, ARMS/Kidins220 interacts with the neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, which have many essential roles in the nervous system such as neuronal survival, neuronal differentiation, dendrites and synapse developments. Recent studies have reported that this protein yields sustained signal of the mitogen-activated protein kinase (MAPK) via the CrkL-C3G complex and Rap1, which are both highly expressed in melanoma. This suggests the ARMS is a potential oncogene and is involved in controlling cell cycle. Cell proliferation is tightly controlled by cyclins and cyclin-dependent kinase (CDKs) in G1, S, M, and G2 phases. First, we observed that knockdown of ARMS/Kidins220 inhibited mouse neuroblastoma cell proliferation and resulted in a slowdown of cell cycle during G1 phase by reductions of cyclin D1 and CDK4. In addition, these decrease in cyclin D1 and CDK4 protein levels cause a subsequent reduction of pRb hyperphosphorylation which occurs at the G1/S phase transition. Moreover, we observed an upregulation of p21, a CDK4 inhibitor, with downregulation of ARMS/Kidins220. Taken together, these data suggest that p21 inhibits the kinase activity of cyclin D1-CDK4 and prevents the hyperphosphorylation of pRb, resulting in down regulation of cell cycle progression. Therefore, we report that the low ARMS/Kidins220 has a role as a signaling mediator to regulate neuroblastoma proliferation and could be worked as a potential oncogene. Human mesenchymal stem cells (hMSCs) can self-renew and differentiate into multiple cell types, which make them suitable for use in tissue engineering and cell therapy. However, their clinical application is hindered by the need for in vitro culture with growth factors in order for their expansion to reach therapeutically useful levels. Moreover, hMSCs consist of a heterogeneous population that exhibits variable morphology, a limited capacity for self-renewal, and inefficient differentiation. Thus, obtaining purified hMSCs with high potential is an important step toward increasing the efficiency of stem cell therapy. For the purpose of flow-based identification of hMSC subpopulations, optimally designed microfluidic chips were developed based on the hydrodynamic filtration (HDF) principle. In these chips, hydrodynamic effects for passive separation are combined with fluid interaction along a series of channels. Microfluidic chip design parameters resulted from complete analysis of laminar flow for flow fraction and complicated networks of main and multi-branched channels, which were validated for microfluidic cell sorting by HDF to fractionate hMSCs into three size-dependent subpopulations: small rapidly self-renewing (RS, < 25 µm)

medium spindle-shaped (SS, 25–40 µm)

and large flattened (FL, > 40 µm) cells. In our HDF chip, a virtual cut-off width (WC) boundary causes hMSCs to migrate to sidewalls, whereby they enter branch channels in response to specific ratios between main and side flows. Our results showed continuous and rapid sorting of hMSCs into three subpopulations with highly efficient recovery (>86%) and complete purity rates, and without damage to cells. Analysis of surface marker expression revealed that RS and SS cells showed high levels of CD73, CD90, and CD105, whereas FL cells did not express CD73. Subsequently, to compare the multipotency of sorted cells, each subpopulation was induced to undergo adipogenic, osteogenic, and Schwann cell (SC) differentiation. Phenotypic and gene marker expression analyses of differentiation indicated that sorted RS and SS subpopulations showed higher potential for adipogenic, osteogenic, and Schwann cell differentiation than the FL subpopulation. Moreover, RS and SS cells displayed significantly increased expression of SC markers and growth factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). Therefore, our results indicate that the microfluidic chip successfully sorted hMSCs based on size and target cells with high multilineage potentials were obtained from hMSCs.