Human Caesarean scar-derived feeder cells: a novel feeder cell type for culturing human pluripotent stem cells without exogenous basic fibroblast growth factor supplementation

2020 ◽  
Vol 32 (9) ◽  
pp. 822
Author(s):  
Wipawee Pavarajarn ◽  
Ruttachuk Rungsiwiwut ◽  
Pranee Numchaisrika ◽  
Pramuan Virutamasen ◽  
Kamthorn Pruksananonda
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Fibroblast Growth ◽  
Feeder Cell ◽  
Feeder Cells ◽  
Cell Type ◽  
Caesarean Scar

In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-β1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.

290 GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM BOVINE FETAL FIBROBLAST CELLS BY DEFINED FACTORS

2011 ◽  
Vol 23 (1) ◽  
pp. 243 ◽  
Author(s):  
H. G. Cao ◽  
Y. Liu ◽  
H. Q. Yin ◽  
X. P. Sun ◽  
Y. S. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Pluripotent Stem Cells ◽  
Leukemia Inhibitory Factor ◽  
Fibroblast Cells ◽  
Fibroblast Growth ◽  
Fetal Fibroblast ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPS) have broad potential applications in drug screening, regenerative medicine, and basic biology, and using iPS as starting materials that have identical properties to embryonic stem cell would probably revolutionize the production of gene-targeted livestock. However, iPS in livestock is still lacking except for in pigs. Instructed by Yamanaka’s idea, in the current study we attempted to generate bovine iPS from fetal fibroblast cells (bFF; from a fetus 2.5 months old after gestation) by using 4 defined transcriptional factors: Oct-4, Sox2, Klf4, and c-Myc. A lentivirus haboring the 4 factors acted as a vehicle to transfect the bFF. One bFF cell line was infected for at least 3 replicates. After transfection, the treated bFF were then cultured in DMEM supplemented with 4 ng mL–1 of basic fibroblast growth factor and 1000 IU mL–1 of leukemia inhibitory factor at 37.5°C, 5% CO2, on the mouse embryonic feeder cells pretreatd with mitomycin C. From Day 16 after the onset of induction, morphology of a few typical spindle-like bFF gradually changed into round, ball-like cells and grew into colonies. Afterward, when these colonies were harvested and subcultured in stem cell medium supplemented with 1000 IU mL–1 of leukemia inhibitory factor and 4 ng mL–1 of basic fibroblast growth factor, new colonies with clear-cut, round edge emerged, and the cells in the colonies had increased nuclear:cytoplasm ratio, kept normal karyotype up to 10 passages, and were alkaline phosphatase staining positive. We also found that the cells exhibited part of the stem cell markers, as evidenced by being Nanog, SSEA1 positive but SSEA3 and TRA-60 negative. Moreover, embryoid bodies could be formed in vitro, and terotoma formation after injection into nude mice also displayed 3 layers. Taken together, we found that 4 lentivirus-mediated, defined transcriptional factors could successfully induce bFF into iPS-like cells. H. G. Cao and Y. Liu contributed equally to this work. The corresponding authors are Y. H. Zhang and X. R. Zhang. This work was supported by NSFC (30800784/c120103, 30700574), 973 (2009CB941004).

The Role of Exogenous Fibroblast Growth Factor-2 on the Reprogramming of Primordial Germ Cells into Pluripotent Stem Cells

Stem Cells ◽  
2006 ◽  
Vol 24 (6) ◽  
pp. 1441-1449 ◽  
Author(s):  
Gabriela Durcova-Hills ◽  
Ian R. Adams ◽  
Sheila C. Barton ◽  
M. Azim Surani ◽  
Anne McLaren
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cells ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth

300 DEVELOPMENTAL STAGE OF BOVINE BLASTOCYSTS AND THE ADDITION OF LEUKEMIA INHIBITORY FACTOR (LIF) AND BASIC FIBROBLAST GROWTH FACTOR (bFGF) DURING CULTURE IN THE ESTABLISHMENT OF EMBRYONIC STEM CELLS-LIKE COLONIES

2013 ◽  
Vol 25 (1) ◽  
pp. 297
Author(s):  
M. D. Guastali ◽  
J. F. Lima Neto ◽  
T. S. Rascado ◽  
D. M. Paschoal ◽  
R. R. D. Maziero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Pluripotent Stem Cells ◽  
Leukemia Inhibitory Factor ◽  
Embryonic Stem ◽  
Fibroblast Growth ◽  
First Passage ◽  
Spontaneous Differentiation

The pluripotent stem cells have the capacity to generate cells of 3 germ layers (ectoderm, mesoderm, and endoderm). The greatest example of pluripotent stem cells are the cells of the inner cell mass (ICM) of the blastocyst, called embryonic stem cells (ESC). Several studies have been conducted to determine the best culture conditions of bovine blastocysts for the establishment of ESCbov. It is known that basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) suppress differentiation in ESC from mice and, but although LIF is important for the growth of ESC in humans, bFGF is not important in preventing differentiation (Prelle et al. 1994 J. Reprod. Fertil.). Because there are no reports in the literature concerning the use of LIF and bFGF in the bovine, the objectives of this study were to use bovine blastocysts in different developmental stages to establish colonies of ESCbov, comparing the action of bFGF and LIF in maintaining the pluripotency of ESCbov during culture. For this, we used bovine blastocysts (IVP) cultured in basal medium with addition of 2.5% FCS; the ICM of early blastocysts, expanded blastocysts, and hatched blastocysts were mechanically removed using 2 insulin needles. The ICM cells were plated in 6 wells containing medium specific for ESC, subdivided into 3 groups: Group 1 (medium + LIF), Group 2 (medium + bFGF), and Group 3 (medium + bFGF + LIF) over a monolayer of mitotically inactivated bovine fibroblasts (20 × 104 cells/1.9 cm2 per well). To confirm the pluripotency of the cells in culture, the colonies were marked with Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 antibodies. The cells were also immunostained for cytokeratin, α fetoprotein, and vimentin to detect spontaneous differentiation. Adhesion and growth of ICM to the monolayer was observed only when blastocysts (n = 160) were used. This adherence began about 2 days after plating. None of the ICM obtained from early blastocysts (n = 300) resulted in colony formation, and the ICM of hatched blastocysts obtained (n = 45) showed a tendency to detach from the monolayer and form cystic embryoid bodies. The use of LIF was fundamental in the establishment of colonies; however, bFGF was not a significant factor in the cultivation of ESC. The colonies presented imunofluorescence for Oct-4, Nanog, SSEA-3, and TRA-1-81, but were negative for SSEA-1, SSEA-4, and TRA-1-60. The cells in primary culture were negative for α-fetoprotein and vimentin, but some colonies showed positive staining for cytokeratin, demonstrating the contamination of the culture with trophoblastic cells. However, after the first passage, cells presented spontaneous differentiation for mesodermal and endodermal tissues. The addition of LIF and bFGF did not reduce spontaneous differentiation. The expanded blastocysts are the most adequate embryos for derivation and establishment of ESC-like colonies in the bovine. The addition of LIF is important for maintaining cells in culture; however, neither LIF nor bFGF prevented spontaneous differentiation after the first passage.

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