Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture

Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.     

人端粒酶反转录酶转染的骨髓间充质干细胞移植治疗大鼠糖尿病

背景：人端粒酶反转录酶是调控增殖及定向分化的首选生长因子之一,具有多重生物学效应。 目的：观察人端粒酶反转录酶表达的骨髓间充质干细胞移植治疗大鼠糖尿病的效果。 方法：体外培养SD大鼠骨髓间充质干细胞,经反转录病毒PLXSN 为载体介导人端粒酶反转录酶基因转染骨髓间充质干细胞,在转染前后用RT-PCR检测骨髓间充质干细胞人端粒酶反转录酶基因的表达。60只雌性SD大鼠中随机取15只作为正常对照组,一次性注射生理盐水,余45只按45 mg/kg的剂量注射链脲霉素建立糖尿病模型后,随机分为3组,分别通过大鼠尾静脉注射移植人端粒酶反转录酶基因转染的骨髓间充质干细胞0.2 mL、骨髓间充质干细胞0.2 mL、生理盐水0.2 mL。 结果与结论：转染48 h后发现,骨髓间充质干细胞有人端粒酶反转录酶mRNA的表达,且重点集中于胞核内。移植后14 d,糖尿病组大鼠空腹血糖维持在较高水平,且高于正常对照组(P < 0.05);与糖尿病组相比,各移植组大鼠空腹血糖水平显著下降(P < 0.05);与骨髓间充质干细胞移植组相比,人端粒酶反转录酶基因转染的骨髓间充质干细胞移植组大鼠空腹血糖水平显著下降(P < 0.05),接近正常对照组水平(P > 0.05)。结果提示人端粒酶反转录酶表达的骨髓间充质干细胞移植能有效治疗大鼠糖尿病。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Coculture of mesenchymal stem cells and respiratory epithelial cells to engineer a human composite respiratory mucosa

In this study, we describe a novel in vitro reconstitution system for tracheal epithelium that could be useful for investigating the cellular and molecular interaction of epithelial and mesenchymal cells. In this system, a Transwell insert was used as a basement membrane on which adult bone marrow mesenchymal stem cells (MSCs) were cultured on the lower side whereas normal human bronchial epithelial (NHBE) cells were cultured on the opposite upper side. Under air-liquid interface conditions, the epithelial cells maintained their capacity to progressively differentiate and form a functional epithelium, leading to the differentiation of mucin-producing cells between days 14 and 21. Analysis of apical secretions showed that mucin production increased over time, with peak secretion on day 21 for NHBE cells alone, whereas mucin secretion by NHBE cells cocultured with MSCs remained constant between days 18 and day 25. This in vitro model of respiratory epithelium, which exhibited morphologic, histologic, and functional features of a tracheal mucosa, could help to understand interactions between mesenchymal and epithelial cells and mechanisms involved in mucus production, inflammation, and airway repair. It might also play an important role in the design of an composite prosthesis for tracheal replacement.     

NT-3基因修饰施万细胞促进神经干细胞体外分化为神经元样细胞

目的：观测神经营养素-3（NT-3）基因修饰施万细胞(SCs)对神经干细胞体外分化为神经元样细胞的影响。方法：神经干细胞(NSCs)分别与NT-3基因修饰SCs（NT-3-SCs）、LacZ基因基因修饰SCs（LacZ-SCs）和SCs在体外共培养，7d后用免疫组化方法观测NSCs的分化并计算其中神经元样细胞的分化率。结果：NSCs在体外可分化为神经元样细胞（NF阳性）和神经胶质样细胞（GFAP阳性），与未基因修饰的SCs相比，NT-3-SCs能更有效地提高神经元样细胞的分化率，而LacZ-SCs与SCs没有明显区别。结论：NT-3-SCs能促进NSCs向神经元样细胞分化。     

成体干细胞的可塑性

继1999年和2000年连续两年被列为年度"人类十大科技进展"后,有关干细胞研究于2007年被再次列为年度十大科学突破之一.干细胞由于具有自我复制、高度增殖和多向分化潜能等特点,已经成为组织细胞发育分化研究、临床医疗应用等领域的重要材料与产品.     

新型化学转染试剂对人脐带间充质干细胞的转染优化

背景：人脐带间充质干细胞的基因修饰技术多种多样,它们在效率、可靠性及安全性方面各有不同。 目的：对比3种新型化学转染试剂在人脐带间充质干细胞中的瞬时转染效率。 方法：采用Fugene HD、Lipofectamine LTX及Attractene分别转染人脐带间充质干细胞,荧光显微镜下观察阳性细胞,流式细胞术分析不同转染试剂的转染效率,锥虫蓝染色观察转染后人脐带间充质干细胞的生存率。 结果与结论：Lipofectamine LTX转染效率最高,显著高于Fugene HD和Attractene[(32.50±2.12)%,(4.30±0.64)%,(1.70±0.08)%,P < 0.05]。Lipofectamine LTX转染后的细胞生存率略低于Fugene HD及Attractene,但差异无显著性意义[(69.8±6.3)%,(92.4±4.2)%,(106.6±3.9)%,P > 0.05]。提示Lipofectamine LTX是人脐带间充质干细胞较为理想的化学转染试剂。     

Elutriated stem cells derived from the adult bone marrow differentiate into insulin-producing cells in vivo and reverse chemical diabetes

An ongoing debate surrounds the existence of stem cells in the adult endowed with capacity to differentiate into multiple lineages. We examined the possibility that adult bone marrow cells participate in recovery from chemical diabetes through neogenesis of insulin-producing cells. Small-sized cells negative for lineage markers derived by counterflow centrifugal elutriation from the bone marrow were transplanted into mice made diabetic with streptozotocin and sublethal irradiation. These cells homed efficiently to the injured islets and contributed to tissue revascularization. Islet-homed CD45-negative donor cells identified by sex chromosomes downregulated GFP, expressed PDX-1 and proinsulin, and converted the hormone precursor to insulin. An estimated 7.6% contribution of newly formed insulin-producing cells to islet cellularity increased serum insulin and stabilized glycemic control starting at 5 weeks post-transplant and persisting for 20 weeks. Newly differentiated cells displayed normal diploid genotype and there was no evidence of fusion between the grafted stem cells or their myeloid progeny and injured β-cells. Considering the extensive functional incorporation of insulin-producing donor cells in the injured islets, we conclude that the adult bone marrow contains a subset of small cells endowed with plastic developmental capacity.     

Neurons and stromal stem cells as targets for polycation-mediated transfection

Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.     

Plasticity of adult stem cells]

Until recently, adults stem cells, defined by their self-renewal and differentiation abilities, were thought to be tissue-specific. This concept has been challenged by bone marrow transplantation experiments in mice, demonstrating generation of cells of different phenotype after transplantation of marrow or muscle cells. The term "plasticity" has been coined to explain this phenomenon which could be due to the persistence in adult tissues, of stem cells with multidifferentiation ability or to the "transdifferentiation" ability of some adult cells committed to differentiation, under the influence of unknown environmental cues. The relationship of the cells at the origin of the stem cells plasticity with a new type of mesodermal cell designed under the term of "multipotent adult progenitor cell" (MAPC) remains to be determined. The discovery of this latter is a major advance in this field as the MAPC have isolated from the adult bone marrow and presents certain characteristics of embryonic stem cells with the demonstration of their totipotency towards many tissues, including hematopoiesis. The discovery of the adult stem cell plasticity phenomenon in general, represents a major change in our concepts of stem and developmental biology and possibly the basis for the development of future cell therapy protocols.     

Comparison of neuron-like cells derived from bone marrow stem cells to those differentiated from adult brain neural stem cells

Bone marrow-derived stem/progenitor cells have been shown by independent investigators to give rise to neural-like cells (neurons and glia) both in vitro and in vivo. The objective of the present study was to determine whether nestin-enriched cells derived from bone marrow can differentiate into cells with the same morphological and functional characteristics as neurons derived from adult brain neurogenic zones. Cell culture methods were used for generation of adult bone marrow and brain stem/progenitor cells and for studying their differentiation into neural-like cells. The proportion of cells expressing neuronal markers was greater in cultures derived from adult hippocampal neural stem cells than in the bone marrow-derived cells, but the electrophysiological and functional characteristics of the cells were similar. Action potentials with electrical characteristics corresponding to those exhibited by adult neural stem cell-derived neurons were recorded from approximately 2.5% of patched neuron-like cells differentiated from bone marrow cells. The active uptake of tritium-labeled neurotransmitters gamma-aminobutyric acid ([(3)H]GABA) and dopamine ([(3)H]DA) was measured in both sets of cultures. [(3)H]GABA uptake, but not [(3)H]DA, was significantly increased in differentiated neurons in both neural stem cell cultures and bone marrow-derived cultures. [(3)H]GABA uptake was greater in differentiated neurons derived from brain neural stem cells. In summary, both the nestin-expressing bone marrow and the adult brain neural stem/progenitors developed into cells with morphological, immunocytochemical, and functional characteristics of neurons. Even though a smaller proportion of neuron-like cells was generated from bone marrow-derived progenitors than from brain-derived neural stem cells, these cells may be useful in the cellular therapy of neurodegenerative diseases and traumatic brain and spinal cord injury.     

A method for single-cell sorting and expansion of genetically modified human embryonic stem cells

Genetic modification of human embryonic stem (hES) cells is essential for studies of gene function and differentiation. The expression of transgenes may direct tissue-specific differentiation and aid in the identification of various differentiated cell types. Stable genomic integration of transgenes is optimal because hES cell differentiation can span several days to weeks and include numerous cell divisions, and establishing homogeneous modified cell lines will facilitate research studies. Herein we provide a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors. Using this method, we have established enhanced green fluorescent protein (eGFP)-expressing hES cell lines that are pluripotent, contain a diploid chromosomal content, and stably express eGFP following more than 2 months of routine culture and in vivo differentiation.     

Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds

Angiogenesis is critical in the early stage of reparative processes and tissue regeneration, but the persistence of a vascular network may interfere with later transformation/maturation in naturally avascular tissues such as articular cartilage. Our supposition is that the timed delivery of an anti-angiogenic factor in cartilage tissue engineering may facilitate the formation of hyaline cartilage by inducing the regression of vascularization. To this end our overall goal is to prepare an off-the-shelf scaffold containing the gene for a potent anti-angiogenic factor. The objective of this study was to investigate the use of a type I/III collagen scaffold for the non-viral transfection of marrow stromal cells (MSCs, also referred to as mesenchymal stem cells) with the plasmid encoding endostatin. Caprine MSCs were transfected by the naked plasmid alone and plasmid incorporated into a cationic lipid complex in three experiments: 1) cells were transfected in monolayer; 2) monolayer-transfected cells were grown in a collagen sponge-like scaffold; and 3) non-transfected cells were grown in a collagen scaffold containing the naked plasmid and endostatin lipoplex. Independent variables were the passage number of the cells and the plasmid loading. The amount of endostatin released by the cells into the medium was measured using an ELISA. The results demonstrated the overexpression of endostatin by MSCs growing in the endostatin lipoplex-supplemented collagen scaffolds. Endostatin released by the cell-seeded scaffolds reached a peak of 13 ng/ml for scaffolds incorporating as little as 20 μg of plasmid, at the 3-day collection period ending 5 days post-seeding. The accumulated endostatin synthesis over a 2-week period began to achieve what may be a therapeutic level. MSCs transfected with the endostatin gene in monolayer continued to express the gene when grown in the collagen scaffolds. The results demonstrate the promise of the non-viral delivery of the gene for this potent anti-angiogenic protein to MSCs via a collagen scaffold.     

Multipotent adult germline stem cells and embryonic stem cells have similar microRNA profiles

Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). microRNAs (miRNAs) belonging to the 290 and 302 miRNA clusters have been previously classified as embryonic stem cell (ESC) specific. Here, we show that these miRNAs generally characterize pluripotent cells. They are expressed not only in ESCs but also in maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, we tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Despite the differences regarding time and extent of differentiation observed between ESCs and maGSCs, expression profiles of both miRNA families showed similarities between these two cell types, suggesting similar underlying mechanisms in maintenance of pluripotency and differentiation. Our results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells can be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. These results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues.     

Anatomical perspectives on adult neural stem cells

The concept of stem cells within the adult brain is not new. However, only recently have scientific techniques become sufficiently advanced to identify them although this remains problematic and the technology is still developing. Nevertheless, it is now generally recognized that stem cells are restricted to two germinal regions within the intact brain. From here they can migrate to specific destinations where they integrate with existing circuitry. Their identity remains controversial but a growing body of evidence suggests it may have an astrocytic phenotype. Within the germinal regions the stem cells are confined to a niche environment and are capable of responding to environmental signals generated locally in an autocrine or paracrine fashion. The niche environment is also modulated by more generalized systemic and physiological activity. These observations are exciting in their own right and form the basis of this review. They are also beginning to alter how we think about neural injury and disease and to impact on the development of novel therapies.     

Nanobiomechanics of repair bone regenerated by genetically modified mesenchymal stem cells

Genetically modified mesenchymal stem cells (MSCs), overexpressing a BMP gene, have been previously shown to be potent inducers of bone regeneration. However, little was known of the chemical and intrinsic nanomechanical properties of this engineered bone. A previous study utilizing microcomputed tomography, back-scattered electron microscopy, energy-dispersive X-ray, nanoindentation, and atomic force microscopy showed that engineered ectopic bone, although similar in chemical composition and topography, demonstrated an elastic modulus range (14.6-22.1 GPa) that was less than that of the native bone (16.6-38.5 GPa). We hypothesized that these results were obtained due to the specific conditions that exist in an intramuscular ectopic implantation site. Here, we implanted MSCs overexpressing BMP-2 gene in an orthotopic site, a nonunion radial bone defect, in mice. The regenerated bone tissue was analyzed using the same methods previously utilized. The samples revealed high similarity between the engineered and native radii in chemical structure and elemental composition. In contrast to the previous study, nanoindentation data showed that, in general, the native bone exhibited a statistically similar elastic modulus values compared to that of the engineered bone, while the hardness was found to be marginally statistically different at 1000 muN and statistically similar at 7000 muN. We hypothesize that external loading, osteogenic cytokines and osteoprogenitors that exist in a fracture site could enhance the maturation of engineered bone derived from BMP-modified MSCs. Further studies should determine whether longer duration periods postimplantation would lead to increased bone adaptation.     

Induction of intervertebral disc-like cells from adult mesenchymal stem cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.