Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.     

A novel silk/PES hybrid nanofibrous scaffold promotes the in vitro proliferation and differentiation of adipose‐derived mesenchymal stem cells into insulin producing cells

Using stem cells to replace the lost beta cells is a hopeful strategy in the treatment of diabetic patients. Furthermore, during stem cell culture and therapy, it is a need to use a substrate to act as a supportive matrix to mimic 3D in vivo microenvironment. Therefore, in this study, human adipose‐derived stem cells were used to differentiate into insulin‐producing cells (IPCs) on a silk/polyethersulfone (PES) scaffold. After exposing to the differentiation media, 2D and 3D (silk/PES) cultured cells were gradually aggregated and formed spherical shaped clusters. The viability of cells was comparable in both 3D and 2D culture. As the results of gene expression assay in both RNA and protein level showed, the differentiation efficiency was higher in 3D culture. Furthermore, ELISA revealed that the release of C‐peptide and insulin was higher in 3D than 2D culture. It seems that silk/PES nanofibrous hybrid scaffold could provide an appropriate matrix to mimic in vivo microenvironment and therefore increases the IPC differentiation potency of stem cells.     

The Effect of Pancreas Islet-Releasing Factors on the Direction of Embryonic Stem Cells Towards Pdx1 Expressing Cells

Diabetes mellitus, which is the result of autoimmune destruction of the insulin-producing β cells, occurs by loss of insulin-secreting capacity. The insufficient source of insulin-producing cells (IPCs) is the major obstacle for using transplantation as diabetes treatment method. The present study suggests a method to form islet-like clusters of IPCs derived from mouse embryonic stem cells (mESCs). This protocol consists of several steps. Before starting this protocol, embryoid bodies (EBs) should be cultured in suspension in conditioned medium of isolated mouse pancreatic islet in combination with activing A to be induced. Then differentiated mESCs were replaced with dishes supplemented with basic fibroblast growth factor (bFGF). Next, bFGF was withdrawn, and cyclopamine and noggin were added. Then the cells were treated with B27, nicotinamide, and islet-conditioned medium for maturation. mESCs, as the control group, were cultured without any treatment. An enhanced expression of pancreatic-specific genes was detected by qRT-PCR and immunofluorescence in the differentiated mESCs. The differentiated mESCsco express other markers of pancreatic islet cells as well as insulin. This method exhibited higher insulin generation and further improvement in IPCs protocol that may result in an unlimited source of ES cells suitable for transplantation. The results indicated that conditioned medium, just as critical components of the stem cell niche associated with other factors, had high potential to differentiate mESCs into IPCs.     

Small molecules that induce cardiomyogenesis in embryonic stem cells

A phenotypic cell-based screen of a large combinatorial chemical library led to the identification of a class of diaminopyrimidine compounds (cardiogenol A-D) which can selectively and efficiently induce mouse embryonic stem cells (ESCs) to differentiate into cardiomyocytes. ESC-derived cardiomyocytes were shown to express multiple cardiac muscle markers, including myosin heavy chain, GATA-4, MEF2, and Nkx2.5, and spontaneously form beating regions. Such small molecules will serve as useful chemical probes to study cardiac muscle differentiation and may ultimately facilitate the therapeutic application of ESCs for cardiac repair.     

A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation

A new micro cell chip which can induce stem cells to differentiate into specific body cell types has been designed and fabricated for tissue engineering. This paper presents the test results of a micro cell stimulator which can provide a new miniaturized tool in cell stimulation, culture and analysis for stem cell research. The micro cell stimulator is designed to apply compressive pressure to the hMSCs (human mesenchymal stem cells) for inducing osteogenesis. The micro cell stimulator is based on the pneumatic actuator with a flexible diaphragm which consists of an air chamber and cell chambers. The hMSCs under cyclic compressive stimulation for one week were observed and assessed by monitoring CD90 (Thy-1), actin, alkaline phosphatase (ALP) and alizarin red expression. The results suggest that cyclic mechanical stimulation is attributed to the different phenomenon of cultured hMSCs in cell proliferation and differentiation. These results are important for the feasibility of the micro cell stimulator to provide the reduction of the necessary quantity of cells, process cost and the increase of the throughput.     

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.     

Restraint of the Differentiation of Mesenchymal Stem Cells by a Nonfouling Zwitterionic Hydrogel

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem‐cell phenotype and multipotency, independent of differentiation‐promoting media, cytoskeletal‐manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem‐cell differentiation and highlights the importance of materials chemistry in maintaining stem‐cell multipotency and controlling differentiation.     

应用原子力显微镜研究淫羊霍苷诱导人脐带间充质干细胞成骨分化

利用碱性磷酸酶(ALP)染色和钙结节(Vonkossa)染色的方法对诱导21 d的淫羊霍苷诱导人脐带间充质干细胞进行鉴定;应用原子力显微镜(AFM)观察淫羊霍苷的形貌和人脐带间充质干细胞诱导0、5、10、15、21 d后的细胞形貌。结果表明,经成骨诱导分化21 d后,ALP染色呈强阳性,Vonkossa染色可见明显钙结节。AFM分析表明,淫羊霍苷在盖玻片上呈分散状分布,在细胞表面上聚集并呈微米域分布。实验发现,由于吸附在细胞表面时,被细胞膜分子包裹,更有利于在细胞表面的吸附,进入细胞内部,细胞表面的淫羊霍苷颗粒较在盖玻片上时增大,由淫羊霍苷颗粒进入细胞后在细胞表面留下一些小孔,可知其通过进入细胞内部诱导成骨分化。分化后,细胞表面有小突触,是由成骨分化后细胞内形成钙结节造成。     

Gelatin/PLLA sponge-like scaffolds allow proliferation and osteogenic differentiation of human mesenchymal stromal cells

Tissue engineering has the potential to supply constructs capable of restoring the normal function of native tissue following injury. Poly(L-lactic acid) (PLLA) scaffolds are amongst the most commonly used biodegradable polymers in tissue engineering and previous studies performed on ovine fibroblasts have showed that addition of gelatin creates a favorable hydrophilic microenvironment for the growth of these cells. The attractiveness of using mesenchymal stromal cells (MSCs) in tissue regeneration is that they are able to differentiate into several lines including osteoblasts. In this study, we investigated the ability of gelatin/PLLA sponges to support the adhesion, proliferation, and osteogenic differentiation of human MSCs isolated from the bone marrow of four donors. [Figure: see text].     

Bmp 2 and Bmp 7 Induce Odonto- And Osteogenesis of Human Tooth Germ Stem Cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.     

Isolation and Characterization of Adipose-derived Mesenchymal Stem Cells (ADSCs) from Cattle

Adipose-derived mesenchymal stem cells (ADSCs) were isolated from the adult adipose tissue of 2-year-old cattle, and then characterized by immunofluorescence and RT-PCR. We found that primary bADSCs could be expanded for 25 passages. Expression of β-integrin, CD44, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 bADSCs were successfully induced to differentiate into osteoblasts and adipocytes. The results indicate the potential for multi-lineage differentiation of bADSCs that may represent an ideal candidate for cellular transplantation therapy.     

Discrimination of primitive endoderm in embryoid bodies by Raman microspectroscopy

Embryoid bodies (EBs), derived from aggregated embryonic stem (ES) cells, are capable of differentiating into all three germ layers, including the endoderm, mesoderm, and ectoderm. The initial stage of EB differentiation is the formation of a primitive endoderm (PE) layer located at the periphery of the aggregate. Raman microspectroscopy was employed to segregate PE cells from undifferentiated ES cells. The Raman spectra of the PE cells of the periphery of EBs, formed upon the withdrawal of leukemia inhibitory factor (LIF), were compared with those of the undifferentiated ES cells of the core of cell aggregates, formed in the presence of LIF. It was noticed that the PE cells have high contents of proteins and low contents of nucleic acids, lipids, and carbohydrates compared with ES cells. Also, we established the presence of another population of PE cells located in the core of the EBs. In addition, we identified some specific Raman markers to distinguish PE cells from ES cells (e.g., I ₁₀₀₃/I ₉₃₇). This is the first study to investigate the PE cells of live EBs and define some Raman markers to distinguish them from undifferentiated ES cells.     

Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application.     

Keratinocyte stem cells and the targets for nonmelanoma skin cancer

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.