The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Protoplasmic astrocytes enhance the ability of neural stem cells to differentiate into neurons in vitro

Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF.     

Adipose tissue-derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor-derived factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.     

The growth inhibitor of African green monkey (BSC-1) cells is transforming growth factors beta 1 and beta 2

The growth inhibitory activity in conditioned medium of African green monkey kidney epithelial (BSC-1) cells that has been shown to arise, at least in part, from transforming growth factor beta 2 (TGF-beta 2) [Hanks, S. K., Armour, R., Baldwin, J. H., Maldonado, F., Spiess, J., & Holley, R. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 79-82] was tested for growth inhibitory activity prior to and following acidification. Similar to TGF-beta 1 from human platelets, the inhibitory activity from BSC-1 cells demonstrated an 8-10-fold stimulation following acidification, showing that the activity was secreted from the cells in latent form. Conditioned medium from BSC-1 cells was collected, acidified, and fractionated by procedures that separate TGF-beta 1 and -2. Biological activity was assayed by using the BSC-1 cell proliferation assay. Two active proteins with properties similar to known TGF-beta 1 and TGF-beta 2 were identified. Identity was confirmed by using immunological and amino acid sequencing techniques. These results were consistent with Northern blot analysis of total BSC-1 RNA, using cDNA probes for TGF-beta 1 and TGF-beta 2, which demonstrated strong signals for both mRNAs. Metabolic labeling in conjunction with two-dimensional gel electrophoresis revealed that the cells secrete approximately 10% TGF-beta 1 and 90% TGF-beta 2.     

Human hair-follicle associated pluripotent (hHAP) stem cells differentiate to cardiac-muscle cells

We have previously demonstrated that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area. HAP stem cells have been previously shown to differentiate to neurons, glial cells, keratinocytes, smooth-muscle cells, melanocytes and cardiac-muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal cord regeneration in mouse models, differentiating to Schwann cells and neurons. In previous studies, we established an efficient protocol for the differentiation of cardiac-muscle cells from mouse HAP stem cells. In the present study, we isolated the upper part of human hair follicles containing human HAP (hHAP) stem cells. The upper parts of human hair follicles were suspended in DMEM containing 10% FBS where they differentiated to cardiac-muscle cells as well as neurons, glial cells, keratinocytes and smooth-muscle cells. This method is appropriate for future use with human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve and spinal cord regeneration in the clinic.     

Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.     

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

Human umbilical cord blood cells transfected with VEGF and L(1)CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis-a novel approach in stem cell therapy

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L₁ cell adhesion molecule (L₁CAM) were used for gene-stem cell therapy of transgenic ^G93^A mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF₁₆₅ (pcDNA-VEGF) and mouse neural L₁ cell adhesion molecule (pcDNA-mL₁CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF + pcDNA-mL₁CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L₁ and VEGF (n = 6), (2) transplantation of un-transfected HUCB cells (n = 5), and (3) control group (n = 5). In first two experimental groups 1 × 10⁶ cells were injected retro-orbitally in pre-symptomatic 22–25-week-old ^G93^A mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L₁CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.     

Down-regulation of Wnt signaling could promote bone marrow-derived mesenchymal stem cells to differentiate into hepatocytes

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be able to differentiate into hepatocytes, but the precise mechanisms controlling this process are unclear. Our aim is try to explore the role of Wnt signaling on the differentiation of BMSCs into hepatocytes. Our study demonstrated that BMSCs could successfully differentiate into hepatocytes under in vitro induction of the tissue extract of damaged liver. The mRNA level of Wnt-1, Wnt-5a, Frizzled1, DSH (disheveled), GSK-3β (glycogen synthase kinase 3 beta) and β-catenin on day 21 when the differentiation direction was determined, was lower than that on days 0, 7, and 11. Furthermore, blocking Wnt-1 signaling by treating BMSCs with Dkk1 could induce BMSCs to express albumin earlier and up-regulation of Wnt signaling by treating BMSCs with Wnt-1 could inhibit BMSCs to differentiate into hepatocytes. Above results indicated that inhibition on Wnt signaling can promote BMSCs to differentiate into hepatocytes.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.