In vitro development of day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles

This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Ham's F12 and Dulbecco's Modified Eagle's Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluorescence to determine the number of nuclei present. Six of seven embryos co-cultured with oviductal exmplants developed to the morula/blastocyst stage, while four of seven embryos co-cultured with trophoblastic vesicles developed to the morula stage. More (P = 0.1) embryos co-cultured with oviductal explants reached the blastocyst stage than embryos co-cultured with trophoblastic vesicles (3 7 vs 0 7 , respectively). The number of cells was higher (P = 0.1) for embryos co-cultured with oviductal explants than for embryos co-cultured with trophoblastic vesicles (162.6 +/- 32 vs 87.3 +/- 28, respectively). The number of cells for embryos co-cultured with either oviductal explants or trophoblastic vesicles appeared to be lower than for embryos matured in vivo that were recovered from the uterus at Day 6 (378, 399, >1000). The co-culture of early equine embryos in a completely defined medium with either trophoblastic vesicles or oviductal explants can support development to at least the morula stage. The co-culture of embryos with oviductal explants resulted in superior development of four-to eight-cell embryos, as indicated by the proportion that reached the blastocyst stage and by the number of cells.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Culture of equine trophoblastic vesicles in vitro

Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Ham's F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were incubated at 37 degrees C in a humidified atmosphere of 90% N(2), 5% O(2), and 5% CO(2) and were examined at 48 and 96 h for the number and diameter of trophoblastic vesicles formed. There was no significant difference (P > 0.1) between HF10 and MEM in the diameter of trophoblastic vesicles at 48 or 96 h of culture. There was, however, a significant increase (P < 0.01) in the number of trophoblastic vesicles per conceptus between 48 and 96 h of culture for HF10 and MEM. The mean diameters of trophoblastic vesicles after 96 h in culture were 396.4 +/- 19.9 mum and 415.0 +/- 12.0 mum for HF10 and MEM, respectively. Histologic sections of trophoblastic vesicles revealed a bilaminar structure consistent with the presence of trophectoderm and endoderm. Squash preparations of trophoblastic vesicles contained cells similar to those recognized in squash preparations of fresh conceptuses. Areas of increased cell density that resembled the inner-cell mass were seen in both fresh trophoblastic vesicles and in sections of trophoblastic vesicles. Equine trophoblastic vesicles may be useful to further the study of early embryonic development and pregnancy recognition in mares.     

Improvement of survival rate of frozen cattle blastocysts after transfer with trophoblastic vesicles

An experiment was conducted to determine if the loss of viability due to deep freezing could be overcome by addition of trophoblastic tissue to the embryo at transfer time. Forty-nine recipient heifers in a cotransfer group each received one frozen blastocyst + two frozen trophoblastic vesicles. The confirmed pregnancy rates by Day 45, 60, and 90 were 73, 61, and 57%, respectively. In a control group of 53 recipients that received only a frozen blastocyst, pregnancy rates for the same periods were 43, 42, and 40%, respectively. The difference between groups was highly significant by Day 45. The addition of trophoblastic vesicles to frozen embryos contributed to luteal maintenance in recipients and likely magnified the intensity of embryonic signals resulting in improved pregnancy rates.     

Co-culture of rat luteal cells with islet cells enhances islet viability and revascularization

Islet cell transplantation is a major treatment strategy for type I diabetes, and has proven to be effective for maintaining glucose homeostasis. However, this treatment requires an extended period of immunosuppression to prevent rejection and recurrent transplantation to maintain function. Thus, to enhance the properties of transplanted islet cells, we examined the effect of the co-culture of luteal cells, which secrete progesterone, on islet cell viability, functionality, and revascularization. It was found that islet viability and functionality were higher in the co-cultured group than in single cultures of islets at 48 and 96 h, in parallel with increased progesterone and vascular endothelial growth factor (VEGF) secretion from luteal cells. In the co-culture groups, VEGF levels at 48 and 96 h and CD31 levels at 48 h were significantly higher than those in the islet groups (p?<?0.001 and p?<?0.05, respectively), and basic fibroblast growth factor (bFGF) levels were increased at 96 h (p?<?0.001). Thus, co-culture with luteal cells may increase islet vascularity by enhancing VEGF and bFGF levels for up to 96 h, which could help to markedly increase the pre-transplantation time to allow for effective immunosuppression therapy. This method may also promote islet cell viability and functionality. Progesterone and angiogenic factors secreted from luteal cells may be responsible for these positive effects.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Co-culture of rabbit one-cell embryos with rabbit oviduct epithelial cells

Summary Rabbit 1-cell embryos were co-cultured with rabbit oviduct epithelial cells (ROEC) to determine if ROEC can enhance embryo development in vitro. Primary ROEC were cultured in serum-free media at 39°C in a 5% CO₂:95% air environment. In experiment 1, 1-cell embryos were co-cultured in Ham's F10 with freshly collected or 4-d-old cultures of ROEC seeded in plastic culture wells or on collagen membranes. One-cell embryos cultured without ROEC served as controls. After 65 h in culture, embryos were stained with Hoechst 33342 to determine the number of cells per embryo. Cell numbers were higher (P<0.035) in all co-culture treatments when compared to controls. Optimal development was obtained by co-culture with 4-d-old ROEC grown on plastic (P<0.003). In experiment 2, Ham's F10, Medium 199, and CZB with glucose medium were compared for their ability to support embryo development in the presence or absence of 4-d-old ROEC growth on plastic. Cell number and the percentage of embryos becoming blastocysts were significantly (P<0.002) higher for embryos cultured in Medium 199 compared to the other media tested. In Medium 199, co-culture with ROEC resulted in only a slight, nonsignificant increase in cell number over culture in Medium 199 alone (110 vs. 96 cells). However, the percentage of embryos reaching the blastocyst stage when co-cultured in Medium 199 with ROEC (49%) was nearly twice (P=0.01) that of embryos in Medium 199 without ROEC (26%). In experiment 3, transfer of embryos cultured in Medium 199 with or without ROEC of 24 or 48 h resulted in no significant differences in posttransfer development. These data indicate a beneficial effect of ROEC on blastogenesis and a salvage effect of ROEC on cell proliferation in embryos grown in a less supportive medium such as Ham's F10. This work was supported by a Multicenter Cooperative Program on Non-HumanIn Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, Bethesda, MD, through Cooperative Agreement HD 21939.     

Glucose utilization by enzymatically-formed trophoblastic vesicles and Day-14 porcine blastocysts

The total glucose metabolism of 48-h spherical trophoblastic vesicles, Day-60 trophoblastic vesicles sections and Day-14 porcine blastocyst sections was measured by the method of O'Fallon and Wright (1). Trophoblastic vesicles were formed by enzyme dispersal in Day-14 porcine blastocysts. Glucose was based on DNA content of the tissue measured by diamino benzoic acid reaction with DNA (2). Slope of the lines (PMoles glucose utilized/4 h x DNA content) was different between Day-14 blastocyst sections and 48 h trophoblastic vesicles (P /= 0.05). Slopes of the lines were identical between 48-h trophoblastic vesicles and Day-60 trophoblastic vesicles sections (P >/= 0.87). Average glucose utilization on a per ng DNA basis was calculated. Day-14 blastocyst sections utilized 0.67 Pmoles glucose/4 h per ng DNA, Day-60 trophoblastic vesicles sections; 0.57; and 48-h sperical trophoblastic vesicles used 0.29. It is hypothesized that the change in glucose utilization between the Day-14 porcine blastocyst and enzymatically formed trophoblastic vesicles may be due to a decrease in metabolism as a consequence of in vitro culture. Further, it is theorized that Day-60 trophoblastic vesicles sections used higher quantities of glucose than 48-h sperical trophoblastic vesicles on a per ng DNA basis due to the increased availability of glucose to the cells of the inner layers, caused by the sectioning of the tissue. The results of this study identify changes in glucose metabolism of enzymatically formed porcine trophoblastic vesicles during culture. It is proposed that enzymatically-formed trophoblastic vesicles be used as a model system for the study of embyro metabolism.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

Co-culture of glomerular epithelial cells and mesangial cells on collagen-gauze-fiber gel

A novel collagen-gauze-fiber gel was created as a scaffold for the co-culture of renal glomerular epithelial cells and mesangial cells at its opposite sides. This gauze-fiber-gel provides a mimic environment like that of renal glomeruli in vivo. The cell morphology, cell growth and cell viability were investigated and the results showed that this novel scaffold maintains cell growth and cell viability without changing cell morphology for more than 3 weeks. Interestingly, glomerular epithelial cells co-cultured with mesangial cells on the gauze-fiber gel resulted in the polarity formation which usually appears on the normal epithelial cells existing at glomerular basement membrane in vivo, but seldom appears on the epithelial cells when cultured in vitro.     

Indomethacin inhibits the uptake of sodium by ovine trophoblastic tissue in vitro

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of ²²sodium ([²²Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF_2α and 13,14-dihydro-15-keto-PGF_2α (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37°C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin mM reduced (P<.01) trophoblastic release (ng/μg DNA) of PGF_2α from

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, reduced PGFM from

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, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [²²Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [²²Na], which is an indirect measure of the transport of water across epithelia, from 3680 ± 1118 to 934 ± 248 cpm/μg DNA (P<.03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of post-mortem electrical stimulation on ovine sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum (SR) vesicles from ovine skeletal muscle were iodinated with the use of immobilized lactoperoxidase to determine the location of proteins in the membrane and to observe any changes resulting from post-mortem electrical stimulation. The labelling pattern of the non-stimulated SR preparations was esentially the same as that observed previously for white muscle SR of rabbit. Most of the membrane protein were labelled, except for the high-affinity calcium-binding protein. Electrical stimulation, however, resulted in an increased labelling of calsequestrin suggesting that this protein is more exposed as a result of such treatment. Certain activities of the adenosinetriphosphatase were affected by electrical stimulation. Both the steady-state concentration of phosphoenzyme and the ATP in equilibrium Pi exchange reaction were significantly reduced by electrical stimulation. It is not known if this alteration in the membrane is responsible for the reduced activity of the SR and thus the greater rate of post-mortem pH in electrically stimulated muscle.     

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles

Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.     

Indomethacin inhibits the uptake of 22sodium by ovine trophoblastic tissue in vitro

Blastocysts from several species synthesize prostaglandins in vitro, but the exact functions of the prostaglandins are unknown. The purpose of this study was to determine if indomethacin, an inhibitor of prostaglandin synthesis, would inhibit the uptake of 22sodium ([22Na]) by ovine trophoblastic tissue. To determine the concentration of indomethacin that would inhibit the synthesis of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM) by blastocysts, blastocysts were collected from ewes 16 days after mating, sliced into pieces approximately 2 mm in length and incubated for 48 h at 37 degrees C in 2 ml of medium containing either 0, 0.2, 0.4, 0.8 or 1.6 mM of indomethacin. Concentrations of indomethacin greater than or equal to 0.2 mM reduced (P less than .01) trophoblastic release (ng/micrograms DNA) of PGF2 alpha from 205 +/- 71.2 to less than or equal to 3.3 +/- 0.2, reduced PGFM from 0.7 +/- 0.1 to less than or equal to 0.17 +/- 0.01, and inhibited formation of trophoblastic vesicles. In a second experiment, blastocysts were recovered from ewes 16 days after mating and pieces of trophoblast were incubated with [22Na] and either 0 or 0.4 mM of indomethacin. Indomethacin reduced the uptake of [22Na], which is an indirect measure of the transport of water across epithelia, from 3680 +/- 1118 to 934 +/- 248 cpm/micrograms DNA (P less than .03) and prevented formation of trophoblastic vesicles. Prostaglandins produced by ovine blastocysts might be involved in controlling uptake of water, which is essential for expansion of blastocysts.     

Acidic vesicles and the uptake of amines by sea urchin eggs

The radioactive amine [¹⁴C]methylamine is accumulated to a great extent by eggs, with kinetics that are dependent upon temperature (Q₁₀ = 5) and sensitive to metabolic inhibitors. Efflux of [¹⁴C]methylamine from eggs (preloaded with tracer concentrations) is increased immediately after fertilization or NH₄Cl activation. Fluorescent amines (9-aminoacridine (9AA) and acridine orange (AO)) are concentrated in small intracellular compartments, presumably vesicles. The possible role of these vesicles in the accumulation of amines by sea urchin eggs and the activation of the metabolism that ensues is discussed.     

Influence of oviductal cells and conditioned medium on porcine gametes

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and beta-mercaptoethanol (25 microM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 microg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.     

Co-culture with TM4 cells enhances the proliferation and migration of rat adipose-derived mesenchymal stem cells with high stemness

The proliferation and migration of mesenchymal stem cells (MSCs) are the efficiency determinants in MSCs transplant therapy. Sertoli cells considered as “nurse cell” possesses the ability to enhance the proliferation and migration of umbilical cord mesenchymal stem cells (UCMSCs). However, no reports about TM4 cells' effect on the proliferation and migration of adipose tissue-derived mesenchymal stem cells (ADSCs) have been found until at present research work. Therefore, this study investigates the effect of TM4 cells on the proliferation and migration of ADSCs. We found that the performance of proliferation and migration of ADSCs were improved significantly while maintaining their stemness and reducing their apoptosis rate. After co-culturing with TM4 cells, the co-cultured ADSCs demonstrated higher proportion of synthetic phase (S) cells and colony-forming units-fibroblastic (CFU-F) number, lower proportion of sub-G1 phase cells and enhanced osteogenic and adipogenic differentiation ability. Moreover, results confirmed the higher multiple proteins involved in cell proliferation and migration including expression of the phospho-Akt, mdm2, pho-CDC2, cyclin D1 CXCR4, MMP-2, as well as phospho-p44 MAPK and phospho-p38 MAPK in co-cultured ADSCs. Furthermore, the process of TM4 cells promoting the proliferation of ADSCs was significantly inhibited by the administration of the PI3K/AKT inhibitor LY294002. Obtained results indicated that TM4 cells through MAPK/ERK1/2, MAPK/p-38 and PI3K/Akt pathways influence the proliferation and migration of ADSCs. These findings indicated that TM4 cells were found effective in promoting stemness and migration of ADSCs, that proves adopted co-culturing technique as an efficient approach to obtain ADSCs in transplantation therapy.