Combined action of stem cell factor, leukemia inhibitory factor, and cAMP on in vitro proliferation of mouse primordial germ cells

1993 ◽  
Vol 35 (2) ◽  
pp. 134-139 ◽  
Author(s):  
Susanna Dolci ◽  
Maurizio Pesce ◽  
Massimo De Felici
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Leukemia Inhibitory Factor ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Inhibitory Factor ◽  
In Vitro Proliferation ◽  
Combined Action

Stem cell factor protects germ cells from apoptosis in vitro

2000 ◽  
Vol 113 (1) ◽  
pp. 161-168 ◽  
Author(s):  
W. Yan ◽  
J. Suominen ◽  
J. Toppari
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Survival Function ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Dna Laddering ◽  
Survival Effect

Stem cell factor (SCF) plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development. However, the function of SCF in the adult testis is poorly described. We have previously shown that, in the presence of SCF, there were more type A spermatogonia incorporating thymidine at stage XII of rat seminiferous tubules cultured in vitro than in the absence of SCF, implying that the increased DNA synthesis might result from enhanced survival of spermatogonia. To explore the potential pro-survival function of SCF during spermatogenesis, the seminiferous tubules from stage XII were cultured in the presence or absence of SCF (100 ng/ml) for 8, 24, 48, and 72 hours, respectively, and apoptosis was analyzed by DNA laddering and in situ 3′-end labeling (ISEL) staining. Surprisingly, not only spermatogonia, but also spermatocytes and spermatids, were protected from apoptosis in the presence of SCF. Apoptosis took place much later and was less severe in the SCF-treated tubules than in the controls. Based on previous studies showing that FSH prevents germ cells from undergoing apoptosis in vitro, and that SCF level is increased dramatically in response to FSH stimulation, we also tested if the pro-survival effect of FSH is mediated through SCF by using a function-blocking monoclonal antibody, ACK-2, to block SCF/c-kit interaction. After 24 hours of blockade, the protective effect of FSH was partially abolished, as manifested by DNA laddering and ISEL analyses. The present study demonstrates that SCF acts as an important survival factor for germ cells in the adult rat testis and FSH pro-survival effect on germ cells is mediated partially through the SCF/c-kit pathway.

Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis)

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1089-1094 ◽  
Author(s):  
M. Pesce ◽  
M.G. Farrace ◽  
M. Piacentini ◽  
S. Dolci ◽  
M. De Felici
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Programmed Cell Death ◽  
Leukemia Inhibitory Factor ◽  
Stem Cell Factor ◽  
Tissue Transglutaminase ◽  
Primordial Germ Cell ◽  
Inhibitory Factor ◽  
High Level

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417–427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530–536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831–838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281–284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4–5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effects of 6-bromoindirubin-3′-oxime on the maintenance of pluripotency of porcine embryonic germ cells in combination with stem cell factor, leukemia inhibitory factor and fibroblast growth factor

Reproduction ◽  
2010 ◽  
Vol 139 (6) ◽  
pp. 1039-1046 ◽  
Author(s):  
Jiang Wen ◽  
Juan Liu ◽  
Guangqi Song ◽  
Limei Liu ◽  
Bo Tang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Germ Cells ◽  
Leukemia Inhibitory Factor ◽  
Stem Cell Factor ◽  
Glycogen Synthase ◽  
Embryoid Bodies ◽  
Inhibitory Factor ◽  
Fibroblast Growth

6-Bromoindirubin-3′-oxime (BIO), which is one of the glycogen synthase kinase 3 inhibitors and a key regulator of numerous signaling pathways, was reported to be capable of maintaining the pluripotency of human and mouse embryonic stem cells. Presently, it is unknown whether BIO can influence the derivation of porcine embryonic germ (EG) cells. In this study, porcine primordial germ cells (PGCs) were isolated from gonads of 24- and 28-day embryos, and were then treated with BIO either individually or in combination with other cytokines (stem cell factor (SCF), leukemia inhibitory factor (LIF), and fibroblast growth factor (FGF); abbreviated as ‘3F’), and the effects of the treatment on the proliferation ability of porcine PGCs at early stage were examined using 5-bromo-2-deoxyuridine (Brdu) immunostaining assay. After continuous culture, the effects on the efficiency of porcine undifferentiated EG cells in the third passage and differentiated EG cells from embryoid bodies were examined as well. The results obtained through the observation of the Brdu-labeled PGCs indicated that BIO in combination with 3F resulted in a significant increase in the mitosis index, and also indicated that the BIO in combination with 3F had a higher efficiency in promoting the formation of porcine EG colony derived from porcine day 24 PGCs than BIO used either individually or in combination with LIF. In addition, BIO in combination with 3F exhibited the apparent anti-differentiation activity by reversing the differentiated EG cells to the undifferentiated status. Our results demonstrate that BIO in combination with SCF, LIF, and FGF could significantly contribute to the establishment of a porcine EG cell colony and maintain the undifferentiated status.

Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor

Zygote ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 271-275 ◽  
Author(s):  
Gabriela Durcova-Hills ◽  
Katja Prelle ◽  
Sigrid Müller ◽  
Miodrag Stojkovic ◽  
Jan Motlik ◽  
...  
Keyword(s):  
Stem Cell ◽  
Primary Culture ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Feeder Cells ◽  
Membrane Bound

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

scholarly journals Effects of the recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 84-95 ◽  
Author(s):  
N Debili ◽  
JM Masse ◽  
A Katz ◽  
J Guichard ◽  
J Breton-Gorius ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Leukemia Inhibitory Factor ◽  
Stem Cell Factor ◽  
Liquid Culture ◽  
Cell Concentration ◽  
Inhibitory Factor ◽  
Concentration Addition ◽  
Potent Inducer ◽  
Cd34 Cells

Using a liquid culture system and human CD34+ marrow cells, we examined the effects of recombinant interleukin (IL)-3, IL-6, stem cell factor (SCF), and leukemia inhibitory factor (LIF) on megakaryocyte (MK) growth, endoreplication, and maturation. MK proliferation, ploidy distribution, and volume were studied by flow cytometry. IL-3 was the only cytokine that, alone, induced a marked increase in MK proliferation. At a high CD34+ cell concentration, addition of IL-6, SCF, and LIF to IL-3--containing medium increased the number of MK (approximately 20%). At a low CD34+ cell concentration, IL-3 alone was a less potent inducer of MK growth, but IL-6, SCF, and their combination had a marked effect, increasing the number of MK by a factor 1.7, 2.9, and 4.4, respectively. These differences may be related to the endogenous release of cytokines in the culture. The effects of these cytokines were subsequently tested on a more mature type of MK progenitor (CD34+ cells isolated after 6 days of incubation in liquid culture). IL-3 remained the most potent cytokine, but IL-6 or SCF alone also increased MK number in comparison to unstimulated cultures. The ploidy distribution of MKs grown with IL-3 was not markedly changed by the addition of the other cytokines, with the exception of SCF, which induced a significant increase in the mean ploidy. However, in all cultures, glycoprotein (GP)IIIa+ 2N and 4N cells were present in large but variable numbers (35% to 75%). The number of these low-ploidy MKs directly correlated with MK proliferation. Therefore, we subsequently explored the absolute number of polyploid MK produced in culture. SCF, IL-6, or their combination, in association with IL-3, increased the number of polyploid MK up to fourfold. In addition, they improved the maturation of MK grown in the presence of IL-3, leading to the synthesis of demarcation membranes and platelet shedding. A similar effect of growth factors on the maturation of day 6 CD34+ cells was observed. We conclude that IL-6 and SCF have a broad range of activities on megakaryocytopoiesis, acting both on the early and late stages. However, the proliferative properties of these cytokines largely predominate in our cultures. Therefore, in the absence of a specific MK regulator, this study further extends the need for a combination of growth factors to maximize megakaryocytopoiesis.

Role of stem cell factor in somatic–germ cell interactions during prenatal oogenesis

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 349-351 ◽  
Author(s):  
Massimo De Felici ◽  
Anna Di Carlo ◽  
Maurizio Pesce
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Molecular Mechanisms ◽  
Primordial Germ Cells ◽  
Significant Progress ◽  
Proliferation And Differentiation ◽  
Complex Events ◽  
Mechanisms Of Migration

During embryogenesis germ cells originate from primordial germ cells (PGCs). The development of mammalian PGCs involves a number of complex events (formation and segregation of PGC precursors, PGC migration and proliferation) which lead to the differentiation of oocytes or prospermatogonia (for a review see De Feliciet al., 1992). During recent years developments in methods for isolation, purification and culture of mouse PGCs have led to significant progress in the understanding of molecular mechanisms of migration, proliferation and differentiation of these cells (for reviews see De Felici, 1994; and De Felici &amp; Pesce, 1994a). In this paper we describe the key role played by stem cell factor (SCF) in PGC development and early folliculogenesis.

Stem Cell Factor Regulation of Apoptosis in Mouse Primordial Germ Cells

1997 ◽  
pp. 19-31 ◽  
Author(s):  
Maurizio Pesce ◽  
Maria Grazia Farrace ◽  
Alessandra Amendola ◽  
Mauro Piacentini ◽  
Massimo De Felici
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells

scholarly journals Primordial germ cells are capable of producing cells of the hematopoietic system in vitro

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 463-472 ◽  
Author(s):  
IN Rich
Keyword(s):  
Stem Cell ◽  
Cell Population ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Methyl Cellulose ◽  
Primitive Streak ◽  
Stem Cell Population ◽  
Hematopoietic Stem ◽  
Hematopoietic System

The identity of the cells giving rise to the hematopoietic system in the mouse embryo are unknown. The results presented here strongly suggest that hematopoietic cells are derived from a nonhematopoietic cell population that has been previously thought to give rise to the germ cells. These cells are called primordial germ cells (PGCs) and can be recognized as large cells showing blebbing and pseudopodial extrusions on their surface. They are alkaline phosphatase (AP) positive and possess a stage-specific embryonic antigen (SSEA-1) on their surface. They represent a small pool of cells in the extraembryonic mesoderm at the base of the allantois in late day-6 embryos. Primordial germ cells from 7.5- and 8.5-day visceral yolk sac and embryo proper form AP+ and SSEA-1+ colonies within 5 days when grown on an embryonic fibroblast feeder cell layer in the presence of leukemia inhibitory factor (LIF), stem cell factor (SCF), and interleukin-3 (IL-3). Individual colonies taken from day-5 cultures can be shown to differentiate into erythroid lineage cells in secondary methyl cellulose culture and produce secondary and tertiary PGCs in the presence of LIF, SCF, and IL-3. Cells taken from the region of the allantois and primitive streak can form colonies on hydrophilic Teflon (DuPont, Wilmington, DE) foils precoated with collagen and fibronectin. The cells from these colonies were then shown to form cobblestone areas on irradiated adult bone marrow stromal layers, indicating that the most primitive in vitro hematopoietic stem cell, the cobblestone-area forming cell (CAFC), was present. PGC colonies were grown in methyl cellulose in the presence of LIF, SCF, and IL-3 for 5 days, and the colonies were removed and passaged 3 times on pretreated extracellular matrix hydrophilic Teflon foils. After each passage, the cells were assayed for their differentiation capacity and PGC content. After the last passage, the number of CAFCs was also determined. It was found that, under these conditions, the PGC population expanded more than 400- fold and also contained CAFCs. It is postulated that the PGC represents a totipotent stem cell population capable of producing a variety of different cell types including cells of the hematopoietic system.

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