Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

<div><p>Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.</p></div

Differential protein expression by undifferentiated ADSCs and ADSCs-derived cells in the absence or presence of Y-27632.

<p>Expression of cMHC (A), cTnI (B), Connexin 45 (C) and RyR2/DHPR (D) in the ADSCs (day 3) and ADSCs-derived cells (day 16, in the absence or presence of Y-27632) by flow cytometer analyses. Right column figures represent results from 3 independent experiments. * <i>P</i><0.05.</p

Expression of cardiac proteins, excitation-contraction coupling proteins and signaling proteins in beating cardiomyocytes.

<p>Expression of MLC-2v(A), cTnT(B) and RyR2/DHPR (C) in the SCR siRNA and ROCK siRNA groups by flow cytometer analyses. (D) Change of JNK and PI3K/Akt from Western Blot analyses. Lane 1 represents undifferentiated ADSCs (day 3), while lanes 2 to 5represent SCR siRNA, ROCKI siRNA, ROCKII siRNA and Both siRNA groups, respectively (day 16). Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p

Changes of ROCK related signaling molecules before or after the differentiation of ADSCs into cardiomyocytes.

<p>(A) Phosphorylation levels of JNK. (<b>B</b>) Phosphorylation level of Akt. Lane 1 represents undifferentiated ADSCs (day 3). Lanes 2 and 3 representbeating cells (day 16) in the absence or presence of Y-27632, respectively. Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p

Immunofluorescence staining of contracting clone for cardiac- and muscle-proteins.

<p>Beating cells were with anti-cMHC (red), anti-α-actin (green), anti-cTnI (red) and anti-Nkx2.5 (red) antibodies. No specific staining was obtained with anti-MyoD (red) and anti-α-SMA (red) antibodies. Nuclei (blue) were stained with Hoechst 33342.</p

Morphological change of actin cytoskeleton during the differentiation of ADSCs.

<p>ADSCs and ADSCs-derived cardiomyocytes were stained with phalloidin for F-actin (green). The white arrows point to stained stress fibers. After 3 days of culture in the absence of Y-27632 (top left panel), stress fibers in cells weres parse. After 7 days of culture in the absence of Y-27632 (top right panel), stress fibersshifted from sparse to dense distribution. The long stress fibers fully occupied the cytoplasm of contractive cells after 16 days of culture in the absence of Y-27632 (middle left and bottom left panels). The contractive cells in the presence of Y-27632 showed much less compact stress fibers in the cytoplasm, in comparison with un-treated cells (after 16 days of culture, middle rightand bottom right panels). The white arrow indicates stress fibers. Nuclei (blue) were stained with Hoechst 33342, Scale bar  = 100 µm.</p

Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.

<p>Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.</p

Morphology and expression of cardiac markers before and after differentiation of ADSCs into cardiomyocytes.

<p>(A) Morphological change of ADSCs into cardiomyocytes in the absence (left panels) or presence (right panels) of ROCK-specific inhibitor Y-27632. After 6 days of culture, spindle shaped cells (black arrow), small round cells (gray arrow) and small tube cells (white arrow) appeared in the top panel, with no beating. After 12 days, single contractive cells (white arrow) were shown in the center panel; After 18dyas, myotube-like structure formed a cohesive network in the bottom panels and they began to contract synchronously. Scale bars  = 100 µm. (B) Expression of cardiac genes was determined by RT-PCR before (lane 1 for undifferentiated ADSCs) and after (lane 2 for beating cells) differentiation of ADSCs into cardiomyocytes. Rat heart tissue was used as a positive control (lane 4), with no cDNA as a negative control (lane 3). Expression of α-cardiac actin, α-MHC, β-MHC, ANP, cTnT, GATA4 and MEF-2C mRNA was higher in ADSCs-derived beating cells than in undifferentiated ADSCs.</p

Table_3_Cellular complexity of the peripheral nervous system: Insights from single-cell resolution.docx

Single-cell RNA sequencing allows the division of cell populations, offers precise transcriptional profiling of individual cells, and fundamentally advances the comprehension of cellular diversity. In the peripheral nervous system (PNS), the application of single-cell RNA sequencing identifies multiple types of cells, including neurons, glial cells, ependymal cells, immune cells, and vascular cells. Sub-types of neurons and glial cells have further been recognized in nerve tissues, especially tissues in different physiological and pathological states. In the current article, we compile the heterogeneities of cells that have been reported in the PNS and describe cellular variability during development and regeneration. The discovery of the architecture of peripheral nerves benefits the understanding of the cellular complexity of the PNS and provides a considerable cellular basis for future genetic manipulation.</p

Polyelectrolyte-Based Platforms for the Delivery of Peptides and Proteins

The use of peptides and proteins in the pharmaceutical field has increased dramatically over recent years. They have been especially relevant to advances in the treatment of cancer, rheumatoid arthritis, leukemia, and cardiovascular, ophthalmological, metabolic, and infectious diseases. Despite the great potential of peptides and proteins, their use in pharmaceuticals has failed to reach its full potential because of some outstanding challenges. They are unstable under storage conditions and in biological milieus, and their high molecular weight limits permeation through biological membranes. A variety of delivery systems have been investigated to overcome these limitations. Polyelectrolytes (PEs) are molecules that bear multiple negative or positive charges. These molecules play an important role in various platforms relating to the delivery of peptide/protein-based drugs and subunit vaccines. The most commonly utilized PEs include chitosan, alginate, chondroitin sulfate, and poly­(γ-glutamic acid). PE-based delivery systems, such as polyelectrolyte complexes (PECs), PE-coated nanocarriers, and PE multilayers, were designed to protect peptides and proteins from degradation and facilitate their absorption. These delivery systems are especially effective when administered orally or intranasally. This review emphasizes the important role of PEs and PE-based delivery vehicles in peptide/protein-based drugs and vaccines