148 INDUCTION OF HYPOMETHYLATION BY EPIGENETIC ALTERATION OF SOMATIC NUCLEI IN CLONED BOVINE BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 182
Author(s):  
G. Wee ◽  
J.-J. Shim ◽  
B.-S. Song ◽  
J.-S. Kim ◽  
D.-B. Koo ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Trichostatin A ◽  
Methylation Status ◽  
Specific Inhibitor ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Centromeric Heterochromatin ◽  
Epigenetic Alteration ◽  
Donor Cells ◽  
Cloned Bovine

Epigenetic reprogramming such as DNA methylation is incomplete in cloned embryos during early development as compared with normal embryos. The increased methylation levels of cloned bovine blastocysts are showed in centromeric heterochromatin. The aim of the present study was to investigate the change of methylation state by treatment of trichostatin A (TSA), a specific inhibitor of histone deacetylase in somatic donor nuclei and cloned blastocyst reconstructed with TSA-treated cells or nontreated cells. Bovine ear skin fibroblast cells (bESF) were used as donor cell and treated with TSA for 60 h at a final concentration of 1 �M. To methylation analysis of satellite I as specific DNA sequence, genomic DNA from 7 � 104 cells and a blastocyst were isolated, and then the genomic DNA was analyzed by bisulfite sequencing. The reduction of HDAC1, 2 and Dnmt family such as Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L after TSA treatment were shown by Western blot in bESF, but histone acetyltransferases such as Tip60 and HAT1 were not changed. Satellite I DNA in nontreated cells was highly methylated in CpG sequences, whereas methylation level of TSA-treated cells was significantly decreased (64 vs. 48%, P < 0.05). After nuclear transfer using normal or altered donor cells, methylation levels of satellite were measured at the blastocyst stage of NT and TSA-NT embryos as compared with IVF embryos. In nontreated NT blastocysts, methylation levels were significantly higher than IVF blastocysts (66 vs. 29%, P < 0.05) and were similar to that of nontreated bESF cells. The reduction of methylation levels in TSA-NT blastocysts were showed and were significantly lower than NT blastocyst derived with nontreated cells (37 vs. 66%, P < 0.05), but no significant differences were found between TSA-NT and IVF blastocysts. Also, the levels of methylation were similar to that of TSA-treated donor cells. In blatocyst formation, TSA-NT embryos were improved significantly compared with NT or IVF embryos (45.9 vs. 31.7 or 28%, P < 0.05). These results demonstrated that somatic methylation status after epigenetic alteration affect in early cloned embryo development, suggesting epigenetic control may help to solve of inherent problems in cloning.

scholarly journals Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

scholarly journals The Effects of 5-Aza-2′- Deoxycytidine and Trichostatin A on Gene Expression and DNA Methylation Status in Cloned Bovine Blastocysts

2011 ◽  
Vol 13 (4) ◽  
pp. 297-306 ◽  
Author(s):  
Yongsheng Wang ◽  
Jianmin Su ◽  
Lijun Wang ◽  
Wenbing Xu ◽  
Fusheng Quan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Trichostatin A ◽  
Methylation Status ◽  
Cloned Bovine

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

6 THE EFFECTS OF DEPLETING DONOR CELL MITOCHONDRIAL DNA ON CATTLE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2016 ◽  
Vol 28 (2) ◽  
pp. 132 ◽  
Author(s):  
K. Srirattana ◽  
J. C. St. John
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Embryo Development ◽  
Copy Number ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Donor Cell ◽  
Mtdna Copy Number ◽  
Donor Cells

Although somatic cell nuclear transfer (SCNT) is a valuable tool for producing animals for agricultural and research purposes, the resultant mixing of mitochondrial DNA (mtDNA) from the donor cell and recipient oocyte (heteroplasmy) affects embryo development and offspring survival and health. The aim of this study was to determine the effects of depleting donor cells of their mtDNA before SCNT on embryo development. mtDNA was depleted from cattle fibroblasts using 2′,3′-dideoxycytidine. mtDNA copy number in cells depleted for 30 days (0.85 ± 0.05) was significantly decreased when compared with nondepleted cells (150.12 ± 29.90; P < 0.0001, ANOVA). Moreover, mtDNA copy number in depleted cells could not be replenished after depletion for 30 days. Depleted cells and nondepleted cells were used as donor cells for SCNT. Somatic cell nuclear transfer embryos were produced by electrofusion of a single donor cell with an enucleated cow oocyte. Reconstructed oocytes were chemically activated and cultured for 7 days (nontreated embryos). Another cohort of embryos was treated with Trichostatin A (TSA), to enhance reprogramming, by activating reconstructed oocytes and culturing them in the presence of 50 nM TSA for up to 10 h. The embryos were then cultured in the absence of TSA. In nontreated groups, the fusion rates of depleted cells (78.0 ± 0.8%) were significantly lower than those of nondepleted cells (92.1 ± 1.4%; P < 0.05). No positive effect on fusion rates was found after TSA treatment. The blastocyst rate for SCNT embryos derived from depleted cells (18.7 ± 4.9%) was significantly lower than the nondepleted group (32.5 ± 3.1%; P < 0.05). Trichostatin A treatment increased blastocyst rates for SCNT embryos derived from depleted cells (32.5 ± 5.3%) to levels equivalent to those of nondepleted cells but did not have any beneficial effect on SCNT embryos derived from nondepleted cells. We have analysed blastocysts for the presence of donor cell mtDNA by high resolution melting analysis. Four out of 10 SCNT blastocysts derived from nondepleted cells were heteroplasmic, whereas others had no donor cell mtDNA. However, all 10 analysed SCNT blastocysts derived from depleted cells were homoplasmic as they harboured only oocyte mtDNA. From RNA sequencing results, TSA treatment of SCNT blastocysts derived from depleted cells increased the expression of key developmental transcription regulators and decreased expression of the mtDNA-specific replication factors, which is essential for embryo development. In conclusion, homoplasmic SCNT embryos were successfully produced by using mtDNA depleted donor cells. Trichostatin A treatment enhanced nuclear reprogramming efficiency in SCNT embryos derived from depleted cells. This work was supported by MitoStock Pty. Ltd., Australia.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 134
Author(s):  
P. Q. Cong ◽  
E. S. Song ◽  
E. S. Kim ◽  
Z. H. Li ◽  
Y. J. Yi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pulse Number ◽  
Donor Cells ◽  
First Polar Body ◽  
Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

In vitro ageing of pig oocytes: effects of the histone deacetylase inhibitor trichostatin A

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 145-152 ◽  
Author(s):  
M. Ješeta ◽  
J. Petr ◽  
T. Krejčová ◽  
E. Chmelíková ◽  
F. Jílek
Keyword(s):  
Histone Deacetylase ◽  
Trichostatin A ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Parthenogenetic Activation ◽  
Parthenogenetic Development ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Developmental Capacity ◽  
In Vitro Ageing

SummaryAfter in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.

scholarly journals Enhancement of in Vitro Developmental Outcome of Cloned Goat Embryos After Epigenetic Modulation of Somatic Cell-Inherited Nuclear Genome with Trichostatin A

2020 ◽  
Vol 20 (1) ◽  
pp. 97-108
Author(s):  
Maria Skrzyszowska ◽  
Marcin Samiec
Keyword(s):  
Somatic Cell ◽  
Trichostatin A ◽  
Nuclear Genome ◽  
Donor Cell ◽  
Group Iv ◽  
Control Group ◽  
Group I ◽  
Donor Cells ◽  
Group Ii

AbstractIn this study, the effect of trichostatin A (TSA)-mediated epigenomic modulation of nuclear donor cells on the in vitro developmental potential of caprine somatic cell cloned embryos was examined. The enucleated ex vivo-matured oocytes were subzonally injected with adult ear skin-derived fibroblast cells exposed or not exposed to TSA (at a concentration of 50 nM). The experiment was designed on the basis of three different approaches to TSA-dependent modulation of donor cell-descended genome: before being used for somatic cell nuclear transfer/SCNT (Group I); immediately after activation of nuclear-transferred (NT) oocytes (Group II); or combined treatment both before being used for SCNT and after activation of NT oocytes (Group III). In the control Group IV, donor cell nuclei have not been treated with TSA at any stage of the experimental design. In TSA-treated Groups I and II and untreated Group IV, cleavage activities of cloned embryos were at the similar levels (80.6%, 79.8% and 77.1%, respectively). But, significant difference was observed between Groups III and IV (85.3 vs. 77.1%). Moreover, in the experimental Groups I and III, the percentages of cloned embryos that reached the blastocyst stages remarkably increased as compared to those noticed in the control Group IV (31.2% vs. 36.7% vs. 18.9%, respectively). In turn, among embryos assigned to Group II, blastocyst formation rate was only slightly higher than that in the control Group IV, but the differences were not statistically significant (25.8% vs. 18.9%). To sum up, TSA-based epigenomic modulation of somatic cell-inherited nuclear genome gave rise to increased competences of caprine cloned embryos to complete their development to blastocyst stages. In particular, sequential TSA-mediated modulation of both nuclear donor cells and activated NT oocytes led to improvement in the blastocyst yields of cloned goat embryos, which can result from enhanced donor cell nuclear reprogrammability.

16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
Z. Mei-Ling ◽  
Z. Yun-Hai ◽  
T. Yong ◽  
L. Ya ◽  
C. Hong-Guo ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Donor Cell ◽  
Developmental Competence ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Different Origins ◽  
Vitro Development

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins

39 IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 126
Author(s):  
Y. Kaedei ◽  
A. Fujiwara ◽  
F. Tanihara ◽  
Z. Namula ◽  
V. L. Vien ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Donor Cells ◽  
Chi Square Analysis ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.

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