The generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed into a pluripotent state by introducing defined transcription factors. iPSCs show almost identical properties in self-renewal and pluripotency, and can circumvent ethical concerns because they do not use embryonic materials. Therefore, iPSCs from a patient's somatic cells have great potential in studying drug development and regenerative medicine. Several human disease models have already been established using patient-specific iPSCs from Parkinson's disease and familial dysautonomia. Moreover, the correction of genetic defects by homologous recombination has already been accomplished with Fanconi anemia patient-specific iPSCs. However, the generation of patient-specific iPSCs for clinical application requires alternative strategies, because genome-integrating viral vectors may raise tumorigenic risk after transplantation. Moreover, the use of iPSCs for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Studies on the mechanism underlying the reprogramming and on establishment of non-integration methods contribute evidence toward resolving the safety concerns associated with iPSCs. Small molecules involved in the epigenetic modification and signaling pathway not only improve reprogramming efficiencies, but also bypass the addition of certain reprogramming factors. However, reprogramming somatic cells purely by small molecule treatment still remains a challenge. Here, we review recent progress made by the use of transcription factors and small molecules that can either replace reprogramming factors or enhance reprogramming efficiency. We also discuss the progress that has been made in the rapidly moving iPSC field, with an emphasis on understanding the mechanisms of cellular reprogramming and its potential application to cell therapy.
Adult ; Dysautonomia, Familial ; Epigenomics ; Fanconi Anemia ; Homologous Recombination ; Humans ; Induced Pluripotent Stem Cells ; Parkinson Disease ; Regenerative Medicine ; Tissue Therapy ; Transcription Factors ; Transplants

Objective: Exploiting their ability to differentiate into mesenchymal lineages like cartilage, bone, fat, and muscle, and to elicit paracrine effects, mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders. One of accessible sources of MSC is the samples used for Papanicolaou (Pap) test, which is a cervical screening method for detecting potentially pre-cancerous and cancerous alterations in the cervical cells and to diagnose genetic abnormalities in fetuses. This study aimed to identify and isolate the stem cells from Pap smear samples collected from pregnant women, and to trace the origin of these cells to maternal or fetal tissue, and characterize their stem cell properties. Methods: To investigate the possibility and efficiency of establishing MSC lines from the Pap smear samples, we were able to establish 6 cell lines from Pap smear samples from 60 pregnant women at different stages of gestation. Results: The 3 cell lines randomly selected among the 6 established in this study, displayed high proliferation rates, several characteristics of MSCs, and the capacity to differentiate into adipocytes, osteocytes, and chondrocytes. Our study identified that the stem cell lines obtainable from Pap smear sampling were uterine cervical stromal cells (UCSCs) and had 10% efficiency of establishment. Conclusion Despite their low efficiency of establishment, human UCSCs from Pap smear samples can become a simple, safe, low-cost, and donor-specific source of MSCs for stem cell therapy and regenerative medicine.

Objective: Exploiting their ability to differentiate into mesenchymal lineages like cartilage, bone, fat, and muscle, and to elicit paracrine effects, mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders. One of accessible sources of MSC is the samples used for Papanicolaou (Pap) test, which is a cervical screening method for detecting potentially pre-cancerous and cancerous alterations in the cervical cells and to diagnose genetic abnormalities in fetuses. This study aimed to identify and isolate the stem cells from Pap smear samples collected from pregnant women, and to trace the origin of these cells to maternal or fetal tissue, and characterize their stem cell properties. Methods: To investigate the possibility and efficiency of establishing MSC lines from the Pap smear samples, we were able to establish 6 cell lines from Pap smear samples from 60 pregnant women at different stages of gestation. Results: The 3 cell lines randomly selected among the 6 established in this study, displayed high proliferation rates, several characteristics of MSCs, and the capacity to differentiate into adipocytes, osteocytes, and chondrocytes. Our study identified that the stem cell lines obtainable from Pap smear sampling were uterine cervical stromal cells (UCSCs) and had 10% efficiency of establishment. Conclusion Despite their low efficiency of establishment, human UCSCs from Pap smear samples can become a simple, safe, low-cost, and donor-specific source of MSCs for stem cell therapy and regenerative medicine.

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.
Animals ; Annexin A5/metabolism ; *Apoptosis ; Blotting, Western ; Cell Fractionation ; Cell Line ; Comparative Study ; Cytochrome c Group/metabolism ; Cytosol/chemistry ; DNA Fragmentation ; Enzyme Activation ; Gene Expression Regulation, Viral ; Hela Cells ; Humans ; Influenza A virus/*physiology ; Kinetics ; Mitochondria/metabolism/*physiology ; Precipitin Tests ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Swine ; bcl-2-Associated X Protein/genetics/metabolism

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.
Animals ; Catalytic Domain/genetics ; *Cell Aging/genetics ; Cell Line, Transformed ; Cell Transformation, Neoplastic ; Dogs ; Embryo/cytology ; Fibroblasts/*cytology/metabolism ; Gene Expression ; Protein p16/genetics ; Protein p53/genetics ; RNA, Messenger/analysis/metabolism ; Research Support, Non-U.S. Gov't ; Telomerase/genetics/metabolism ; ras Proteins/genetics/metabolism

Human embryonic stem cells (hESCs) need feeder cells for their maintenance in an undifferentiated state. In conventional culture systems, mouse embryonic fibroblasts (MEFs) serve as feeder cells to maintain hESCs. However, the use of MEFs elevates the risk of transmitting mouse pathogens and thus limits the potential of hESCs in cell replacement therapy. Consequently, the use of human feeder cells would be an important step forward in this in vitro technology. To address this issue, we used fibroblast-like cells differentiated from the Miz-hES6 hESC line (Diff (Miz-hES6)) as feeder cells to support the in vitro growth of three hESC lines. Immunofluorescence microscopy and reverse transcription-PCR assessing the expression of undifferentiated hESC markers revealed all three hESC lines were maintained in an undifferentiated state. In vitro proliferation proceeded as efficiently as when the hESCs were cultured on MEFS. Moreover, karyotype analysis revealed the chromosomal normality of the hESC lines and the Diff (Miz-hES6) feeders themselves after even 50 passages. Furthermore, the hESC lines maintained their pluripotency since they remained capable of forming embryoid bodies (EBs) in vitro. Thus, hESC-derived fibroblast-like cells successfully support in vitro hESC propagation.
Biological Markers/analysis ; Cell Culture Techniques/*methods ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Embryo/*cytology ; Fibroblasts/cytology ; Humans ; Karyotyping ; Pluripotent Stem Cells/cytology ; Research Support, Non-U.S. Gov't ; Stem Cells/*cytology ; Time Factors

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology

BACKGROUND: Immunoglobulin A (IgA) nephropathy (IgAN) is one of an important cause of progressive kidney disease and occurs when IgA settles in the kidney resulted in disrupts kidney’s ability to filter waste and excess water.Hydrogels are promising material for medical applications owing to their excellent adaptability and filling ability. Herein, we proposed a hyaluronic acid/gelatin (CHO-HA/Gel-NH2 ) bioactive hydrogel as a cell carrier for therapeutic kidney regeneration in IgAN. METHODS: CHO-HA/Gel-NH2 hydrogel was fabricated by Schiff-base reaction without any additional crosslinking agents. The hydrogel concentrations and ratios were evaluated to enhance adequate mechanical properties and biocompatibility for further in vivo study. High serum IgA ddY mice kidneys were treated with human urine-derived renal progenitor cells encapsulated in the hydrogel to investigate the improvement of IgA nephropathy and kidney regeneration. RESULTS: The stiffness of the hydrogel was significantly enhanced and could be modulated by altering the concentrations and ratios of hydrogel. CHO-HA/Gel-NH2 at a ratio of 3/7 provided a promising milieu for cells viability and cells proliferation. From week four onwards, there was a significant reduction in blood urea nitrogen and serum creatinine level in Cell/Gel group, as well as well-organized glomeruli and tubules. Moreover, the expression of pro-inflammatory and profibrotic molecules significantly decreased in the Gel/Cell group, whereas anti-inflammatory gene expression was elevated compared to the Cell group. CONCLUSION Based on in vivo studies, the renal regenerative ability of the progenitor cells could be further increased by this hydrogel system.

BACKGROUND: Immunoglobulin A (IgA) nephropathy (IgAN) is one of an important cause of progressive kidney disease and occurs when IgA settles in the kidney resulted in disrupts kidney’s ability to filter waste and excess water.Hydrogels are promising material for medical applications owing to their excellent adaptability and filling ability. Herein, we proposed a hyaluronic acid/gelatin (CHO-HA/Gel-NH2 ) bioactive hydrogel as a cell carrier for therapeutic kidney regeneration in IgAN. METHODS: CHO-HA/Gel-NH2 hydrogel was fabricated by Schiff-base reaction without any additional crosslinking agents. The hydrogel concentrations and ratios were evaluated to enhance adequate mechanical properties and biocompatibility for further in vivo study. High serum IgA ddY mice kidneys were treated with human urine-derived renal progenitor cells encapsulated in the hydrogel to investigate the improvement of IgA nephropathy and kidney regeneration. RESULTS: The stiffness of the hydrogel was significantly enhanced and could be modulated by altering the concentrations and ratios of hydrogel. CHO-HA/Gel-NH2 at a ratio of 3/7 provided a promising milieu for cells viability and cells proliferation. From week four onwards, there was a significant reduction in blood urea nitrogen and serum creatinine level in Cell/Gel group, as well as well-organized glomeruli and tubules. Moreover, the expression of pro-inflammatory and profibrotic molecules significantly decreased in the Gel/Cell group, whereas anti-inflammatory gene expression was elevated compared to the Cell group. CONCLUSION Based on in vivo studies, the renal regenerative ability of the progenitor cells could be further increased by this hydrogel system.