Three-Dimensional Imaging in Stem Cell-Based Researches.

Stem cells have an important role in regenerative therapies, developmental biology studies and drug screening. Basic and translational research in stem cell technology needs more detailed imaging techniques. The possibility of cell-based therapeutic strategies has been validated in the stem cell field over recent years, a more detailed characterization of the properties of stem cells is needed for connectomics of large assemblies and structural analyses of these cells. The aim of stem cell imaging is the characterization of differentiation state, cellular function, purity and cell location. Recent progress in stem cell imaging field has included ultrasound-based technique to study living stem cells and florescence microscopy-based technique to investigate stem cell three-dimensional (3D) structures. Here, we summarized the fundamental characteristics of stem cells via 3D imaging methods and also discussed the emerging literatures on 3D imaging in stem cell research and the applications of both classical 2D imaging techniques and 3D methods on stem cells biology.

Efficient and cost-effective generation of hepatocyte-like cells through microparticle-mediated delivery of growth factors in a 3D culture of human pluripotent stem cells.

Biomedical application of human pluripotent stem cell-derived hepatocyte-like cells (hPSC-HLCs) relies on efficient large-scale differentiation, which is commonly performed by a suspension culture of three-dimensional (3D) multicellular spheroids in bioreactors. However, this approach requires large amounts of growth factors (GFs) and the need to overcome limited diffusional transport posed by the inherent 3D structure of hPSC spheroids. Here, we have hypothesized that localized delivery of GFs by incorporation of GF-laden degradable polymeric microparticles (MPs) within the hPSC spheroids would circumvent such limitations. In this study, GFs for hepatocytic differentiation were encapsulated in gelatin-coated poly (l-lactic acid)/poly (DL-lactic-co-glycolic acid) (PLLA/PLGA) MPs which were subsequently incorporated into the hPSC spheroids. Gene expression analyses demonstrated that MP delivery of the GFs resulted in similar expression levels of hepatocytic markers despite the use of 10-fold less total GFs. The differentiated HLCs in the MP group exhibited ultrastructure and functional characteristics comparable with the conventional soluble GF group. The generated HLCs in the MP group were successfully engrafted in an acute liver injury mouse model and maintained hepatocytic function after implantation. These results suggested that sustained and localized delivery of GFs using MPs might offer a novel approach towards scalable technologies for hepatocytic differentiation and engineer a better 3D microenvironment for cells.

Developing a Cost-Effective and Scalable Production of Human Hepatic Competent Endoderm from Size-Controlled Pluripotent Stem Cell Aggregates.

Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for potential research and therapeutic applications. However, current protocols for scalable and integrated differentiation of hPSCs limited due to high cost of growth factors and technical challenges. Here, hPSCs aggregates primed with 6 and 12 µM of CHIR99021 (CHIR), a Wnt agonist, in combination with different concentrations of high cost Activin A (10, 25, 50, 100 ng/mL). We sought to determine the appropriate treatment duration for efficient and cost-effective differentiation protocol for foregut definitive endoderm production in a dynamic suspension culture. Afterward, we evaluated the impact of the initial hPSC aggregate sizes (small: 86 ± 18 µm; medium: 142 ± 32 µm; large: 214 ± 34 µm) as critical bioprocess parameter on differentiation efficacy at the beginning of induction. The results indicated that 1-day priming of hPSCs as 3D aggregates (hPSpheres) with 6 µM CHIR followed by treatment with a low concentration of Activin (10 ng/mL) for 2 days resulted in efficient differentiation to definitive endoderm. This finding confirmed by the presence of ≥70% SOX17/FOXA2-double positive cells that highly expressed the anterior endodermal marker HEX. These endodermal cells differentiated efficiently into mature functional hepatocytes [60% albumin (ALB)-positive cells]. The results showed that the initial size of hPSC aggregates significantly impacted on the efficacy of differentiation. The medium sized-hPSpheres resulted in higher productivity and differentiation efficiency for scalable hepatocytes production, whereas small aggregates resulted in significant cell-loss after CHIR treatment and large aggregates had less efficacious endodermal differentiation. Differentiated cells exhibited multiple characteristics of primary hepatocytes as evidenced by expressions of liver-specific markers, indocyanine green and low-density lipoprotein uptake, and glycogen storage. Thus, this platform could be employed for scalable production of hPSC-derived hepatocytes for clinical and drug discovery applications.

Autologous transplantation of mesenchymal stromal cells tends to prevent progress of interstitial fibrosis in a rhesus Macaca mulatta monkey model of chronic kidney disease.

BACKGROUND AIMS: Chronic kidney disease (CKD) attributed to cisplatin is well documented. Mesenchymal stromal cells (MSCs) are proven to be renotropic. Although they have been shown to improve function in CKD and reduce fibrosis in different experimental rodent models, their efficiency in primates is unknown. The present study aimed to evaluate the prevention of CKD and reduction of fibrosis in monkeys treated with MSCs after cisplatin nephrotoxicity. METHODS: We induced CKD in adult rhesus Macaca mulatta monkeys by means of intravenous administration of cisplatin. Autologous MSCs were transplanted by means of intrarenal arterial injections to assess the adverse effects of cisplatin in two CKD models: preventative and stable. Preventative CKD monkeys (n = 3) underwent cell transplantation 4 days after the cisplatin injection. The stable CKD monkeys (n = 2) underwent cell transplantation 6 months after the cisplatin injection. Non-treated (n = 4) and normal saline-injected animals (n = 3) comprised the control and vehicle groups, respectively. We followed the animals for survival rate, serum biochemistry, urine analysis and histopathological indices. RESULTS: In the preventive CKD model, MSC transplantation tended to improve some renal functions but significantly reduced the histopathologic score compared with the vehicle and control groups. In the stable CKD model, MSCs did not ameliorate renal function or pathological score. CONCLUSIONS: These results suggest that MSCs tend to delay progression of CKD and fibrosis but do not reduce established interstitial fibrosis in this unique primate model of cisplatin-induced nephrotoxicity.

Intra-renal arterial injection of autologous bone marrow mesenchymal stromal cells ameliorates cisplatin-induced acute kidney injury in a rhesus Macaque mulatta monkey model.

BACKGROUND: Clinically, acute kidney injury (AKI) is a potentially devastating condition for which no specific therapy improves efficacy of the repair process. Bone marrow mesenchymal stromal cells (BM-MSCs) are proven to be beneficial for the renal repair process after AKI in different experimental rodent models, but their efficacy in large animals and humans remains unknown. This study aims to assess the effect of autologous rhesus Macaque mulatta monkey BM-MSC transplantation in cisplatin-induced AKI. METHODS: We chose a model of AKI induced by intravenous administration of 5 mg/kg cisplatin. BM-MSCs were transplanted through intra-arterial injection. The animals were followed for survival, biochemistry analysis and pathology. RESULTS: Transplantation of 5 × 10(6) cells/kg ameliorated renal function during the first week, as shown by significantly lower serum creatinine and urea values and higher urine creatinine and urea clearance without hyponatremia, hyperkalemia, proteinuria and polyuria up to 84 d compared with the vehicle and control groups. The superparamagnetic iron oxide nanoparticle-labeled cells were found in both the glomeruli and tubules. BM-MSCs markedly accelerated Foxp3+ T-regulatory cells in response to cisplatin-induced damage, as revealed by higher numbers of Foxp3+ cells within the tubuli of these monkeys compared with cisplatin-treated monkeys in the control and vehicle groups. CONCLUSIONS: These data demonstrate that BM-MSCs in this unique large-animal model of cisplatin-induced AKI exhibited recovery and protective properties.

Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.