Progress 07/06/15 to 06/30/20
Outputs
Target Audience:Training of undergraduates and graduate students in cellular and molecular biological research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A doctoral student, Ran Zhou, graduated in 2018 and obtained a postdoctoral position at the Carnegie Institute of Embryology, Baltimore, MD. Two articles were published from her doctoral work. One was published in 2018 electronic/2019 print and the second in 2019 Typically 2 to 3 undergraduates assist in the stem cell research, learning cell culture and molecular/cellular biology techniques, and the development of SOPs. How have the results been disseminated to communities of interest?The results from this research have been presented to the scientific community at annual conferences and in scientic publications. In addition, information is posted on the lab website (Keeferlab.com) and through ResearchGate. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Research was focused on deriving stem cells for felids including embryonic stem cells (ESC) from in vitro produced blastocysts, spermatogonical stem cells from pre-pubertal and adult testicular tissues (SSC), and induced pluripotent stem cells (iPSC) from fibroblasts. 1. Propagation of SSC: Earlier and ongoing studies demonstrated that SSC could be isolated from pre-pubertal and adult cat testicular tissues and propagated for multiple passages (>5) while maintaining expression of SSC markers (PLZF, UCHL1, and OCT4). Established procedures for the derivation, passaging, and characterization of these cells were used to train undergraduates in cellular and molecular biological techniqes. 2. Derivation of ESC-like cells: Our objective was to understand how cytokines influence pluripotency in the cat inner cell mass (ICM) outgrowths. Cat ICM was isolated from in vitro-produced embryos and outgrowths were cultured for up to 6 days with single or combined cytokines. Cell proliferation was enhanced with almost all single growth factors and cytokine combinations. Based on gene expression and presence of NANOG, POU5F1, and Sex-determining region Y box 2 (SOX2) as cell state markers, single growth factors could not maintain similar levels in outgrowths as in the original ICMs, which is different from the response in mouse and human. In our conditions, cytokine combinations involving LIF, GSK3 inhibitor, and MEK inhibitor resulted in the most robust expression levels and allowed single-cell dissociation and propagation. However, further characterization of embryonic cells derived from ICM indicated that the pluripotent state was not fully preserved. The absence of detectable transcripts for BMP2- receptor and SMAD4, and very low levels of LIF-receptor and STAT3 in the cat ICM indicated that pluripotency regulatory machinery appear to be different in the cat from the predominant mouse and human models (Zhou et al. 2019/epub2018). 3. Reprograming of somatic cells (iPSC): Incomplete transgene-silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids-a critical family in biomedical and biodiversity conservation science. In this study doxycycline-inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline-phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription-quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells (Zhou et al. 2019).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhou R, Comizzoli P, Keefer CL. 2019. Endogenous pluripotent factor expression after reprogramming cat fetal fibroblasts using inducible transcription factors. Mol Reprod Dev. 86(11):16711681. doi:10.1002/mrd.23257
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhou R, Wildt D, Keefer C, Comizzoli P. 2019. Combinations of growth factors regulating LIF/STAT3, WNT and FGF2 pathways sustain pluripotency-related proteins in cat embryonic cells. Stem Cells Dev. 28(5):329-340. doi:
10.1089/scd.2018.0109.[1 citation].
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Zhou, Ran. 2018. Derivation of pluripotent stem cells from blastocysts and somatic cells in the domestic cat (Felis Catus). University of Maryland College Park. https://doi.org/10.13016/M2FQ9Q86H
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Undergraduate and graduate student training in cell culture and cellular and molecular techniques. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A doctoral student, Ran Zhou, graduated in 2018 and obtained a postdoctoral position at the Carnigie Institute of Embryology, Baltimore, MD. Two publications should result from her doctoral work. One was published in 2018 electronic/2019 print. Typically 2 to 3 undergraduates assist in the stem cell research, learning cell culture and molecular/cellular biology techniques. How have the results been disseminated to communities of interest?The results from this research have been presented to the scientific community at annual conferences and in scientic publications. In addition, information is posted on the lab website (Keeferlab.com) and through ResearchGate. What do you plan to do during the next reporting period to accomplish the goals?Publish the second manuscript from the graduate student's (Ran Zhou's) dissertation. Continue training undergraduate researchers.
Impacts
What was accomplished under these goals?
Our objective was to understand how cytokines influence pluripotency in the cat inner cell mass (ICM) outgrowths. Cat ICM was isolated from in vitro-produced embryos and outgrowths were cultured for up to 6 days with single or combined cytokines. Cell proliferation was enhanced with almost all single growth factors and cytokine combinations. Based on gene expression and presence of NANOG, POU5F1, and Sex-determining region Y box 2 (SOX2) as cell state markers, single growth factors could not maintain similar levels in outgrowths as in the original ICMs, which is different from the response in mouse and human. In our conditions, cytokine combinations involving LIF, GSK3 inhibitor, and MEK inhibitor resulted in the most robust expression levels and allowed single-cell dissociation and propagation. However, further characterization of embryonic cells derived from ICM indicated that the pluripotent state was not fully preserved. The absence of detectable transcripts forBMP2-receptorandSMAD4, and very low levels ofLIF-receptorandSTAT3in the cat ICM indicated that pluripotency regulatory machinery appear to be different in the cat from the predominant mouse and human models
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhou R, Wildt D, Keefer C, Comizzoli P. 2019. Combinations of growth factors regulating LIF/STAT3, WNT and FGF2 pathways sustain pluripotency-related proteins in cat embryonic cells. Stem Cells Dev. 28(5):329-340. doi: 10.1089/scd.2018.0109.[1 citation].
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Training in cell culture and experimental techniques were provided to two undergraduates, who carried out all the work involved in the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?As this project has been carried out predominantly by graduate (past years) and undergraduate (this year) students, it has provided an excellent vehicle for training students in cell biology and experimental design. The undergraduate currentlyin charge of the project is planning to present a poster at the ANSC Annual Symposium in May 2019. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Spermatogonial stem cells (SSC) may provide an innovative solution for the preservation of genetic diversity in endangered species and propagation of valuable genetics for domestic livestock. For endangered species, the lost of even one juvenileindividual can reduce the genetic diversity of the small pool of animals available for breeding programs. Yetthere is the potential to rescue the genetics if the SSC from young male animals could be collected and propagated in culture asSSC can be transferred into testes ofhost animals for completion of spermatogenesis.However,SSC from animal species other than mouse lose the ability toform sperm with continued culture. In this project, graduate and undergraduate students have modified the in vitro conditions used in the mouse modelto improve the survival of SSC derived fromcat testes obtained from veterinary clinics following routine neutering procedures. Two approaches have been taken; 1)isolation and propagation of SSC using procedures initiallyestablished by a graduate student in our lab, and 2) modification of a testicular tissue culture procedures intially developed in the mouse.Using the first approach, felid cat SSC have been propogated over multiple passages while retaining molecular and morphological characteristics of SSC. Studies are ongoing that will assess the ability of the SSC to form sperm following transfer into a host animal. Findings from studies using the second approachhave demonstrated that this method does not work with felid tissues. The tissue dies within days of initiation of culture. This failure is likely due to the denser structure of cat testes as compared to the mouse. In future studies this approach will be modifed. Testicular material will be dissociated into loose cells and then reconstituted in a 3D gelmatrix. The hypothesis being that by loosening the connective tissue and structure of the felid testicular tissue, nutrients and waste product can more readily enter and exit the tissues.
Publications
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Undergraduate training: ANSC undergraduate students are exposed to the demands of research. Each participates in a portion of the project (collection, isolation, and culture of spermatogonial stem cells). Graduate student training: A doctoral student who has her own research project (isolation, culture, and characterization of feline ESC) has been in charge of training the undergraduates students. Thus she has gained experience in mentoring. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for a graduate student (doctoral) and several undergraduate students (usually one to three per academic year). How have the results been disseminated to communities of interest?Abstract (poster) has been presented at the annual conference of the Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals?Continue to work on cat ESC and initiate cat iPSC study (doctoral student) and continue the SSC project to train undergraduates.
Impacts
What was accomplished under these goals?
1. Graduate student project - feline ESC (R. Zhou): When propagating pluripotent cells from early-stage embryos, the level of dependence on LIF/STAT3 and FGF2/MEK/ERK signaling pathways are species-specific. However, culture conditions that maintain pluripotency in embryonic stem cells (ESC) using various combinations of growth factors (GF; targeting LIF or FGF2 pathways) and inhibitors (targeting WNT/GSK3 or FGF2 pathways) have yet to be determined in the cat model. We previously observed that, in absence of GFs or inhibitors, pluripotency-related proteins NANOG and POU5F1 were decreased in ICM outgrowths at 144 hours of culture. Additionally, presence of SOX2 was decreased at 72 hours. In Experiment 1, in vitro produced (IVP) blastocysts (n=3-5 per treatment) were cultured in the presence of single GF at three concentrations. Cell number (Hoechst staining) and pluripotency marker expression (immunostaining) in the ICM outgrowths were assessed at 144 hr. Supplementations with FGF2 (10 or 20 ng/ml), hLIF (10 or 100 ng/ml) and fLIF (1, 10 or 100 ng/ml) increased (P<0.05, MANOVA) cell number in the outgrowths compared to controls without GF. FGF2 (5 or 10 ng/ml), hLIF (10 ng/ml) and fLIF (100 ng/ml) sustained SOX2 expression at higher (P<0.05) levels compared to outgrowths without GFs. NANOG expression was maintained by FGF2 (5 or 10 ng/ml), hLIF (1 or 10 ng/ml) and fLIF (1, 10 or 100 ng/ml) at higher (P<0.05) levels compared to outgrowths without GFs. FGF2 (5 ng/ml), hLIF (100 ng/ml) and fLIF (1 or 10 ng/ml) maintained POU5F1 at higher (P<0.05) levels compared to outgrowths without GFs. Because no single cytokine maintained all three pluripotency markers to levels comparable to ICMs in cat blastocysts, cytokine combinations affecting multiple pathways were tested. In Experiment 2, 17 combinations were tested using IVP blastocysts (n = 3-5 per treatment). ICM outgrowths were assessed for cell number and pluripotency marker expression at 144 hours. Four combinations (10 ng/ml fLIF + 10 ng/ml hLIF + 5 ng/ml FGF2; 20 ng/ml fLIF + 3 µM GSK3i + 1 µM MEKi; and 10 ng/ml fLIF + 10 ng/ml hLIF + 3 µM GSK3i with or without 1 µM MEKi) sustained SOX2, POU5F1 and NANOG expression at similar (P>0.05, Chi-Square after K-Clustering) levels compared to ICMs in blastocysts. Furthermore, two of these combinations (10 ng/ml fLIF + 10 ng/ml hLIF + 5 ng/ml FGF2 and 10 ng/ml fLIF + 10 ng/ml hLIF + 3 µM GSK3i + 1 µM MEKi) enhanced (P<0.05) cell proliferation compared to outgrowths without GFs. In summary, specific cytokine combinations of fLIF and hLIF with either FGF2 or the 2 inhibitors maintained pluripotency and supported proliferation over a longer interval in vitro than single cytokine supplementation, likely through synergistic activation of multiple pathways. 2. Undergraduate Project (feline SSC): Several undergraduates have been trained to culture SSC, although little progress was made on this project due to their limited time commitment to the project. One undergraduate completed a project in which he attempted to isolate intact testicular extracellular matrix akin to "ghost" hearts. He was able to successfully isolate ghost testes from mouse (extracellular matrix retaining the shape of a testes without contaminating cellular or DNA debris) using the same detergents and treatments used by other labs to obtain organ matrixes, but due to the increased density and size of the cat testes was able only to partially eliminate DNA debris in the cat. Using younger cat testes and longer treatments could overcome this problem.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Zhou R., Comizzoli P., Wildt D.E., Keefer C.L. (2017). Specific combinations of cytokines enhance cell proliferation and expression of pluripotency-related proteins in inner cell mass outgrowths in the domestic cat model. Abstract no. 244, 50th Annual Meeting for the Society for the Study of Reproduction, Washington, DC.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Undergraduate training: ANSC undergraduate students are exposed to the demands of research. Each participates in a portion of the project (collection, isolation, and culture of spermatogonial stem cells). Graduate student training: A doctoral student who has her own research project (isolation, culture, and characterization of feline ESC) has been in charge of training the undergraduates students. Thus she has gained experience in mentoring. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for a graduate student(doctoral) and severalundergraduate students (usually one to three per academic year). How have the results been disseminated to communities of interest?Abstract (poster) have been presented at the annual conference of the Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals?Continue to work on cat ESC and initiate cat iPSC study (doctoral student) and continue to is the SSC project totrainundergraduates.
Impacts
What was accomplished under these goals?
1: Graduate project (R. Zhou, PhD student): ICMS were isolated from expanded in vitro produce blastocysts on day 6 or 7 after fertilization. Cell clusters from inner cell mass were cultured in the absence of cytokines did not maintain pluripotency markers (NANOG, SOX2, OCT4). Subsequent experiments tested the effects of different feeder cells (mouse embryonic fibroblasts (mEFs) and mouse C166 cells and cytokine supplementation. The combination of mEF feeder cells and Lif+2i (MEK inhibitor (1uM) and GSK β-3 inhibitor (3uM)) gave the best result with the formation of ES-like colonies. However, this simple supplementation failed to support sufficient cell proliferation during passaging. This result raised several questions regarding what concentrations and combinations and which pathways (e.g., LIF, BMP, TGFbeta/activing/nodal, and/or FGF) should be stimulated in the cat cells in order to maintain pluripotency. 2. Graduate/undergraduate project: A previous graduate student (L. Vansandt, 2014) had established a system to enrich and maintain feline spermatogonial cells. R. Zhou was able to repeat the established procedures and could maintain frozen/thawed SSC to passage 7 that had similar protein expression (PLZF, UCHL1) pattern as fresh SSC. She trained several undergraduates in the methods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Zhou R., Comizzoli P., Wildt D.E., Keefer C.L. (2016). Defining Conditions That Maintain Pluripotency of Embryonic Stem Cells in Domestic Cats. Abstract no. 332, 49th Annual Meeting for the Society for the Study of Reproduction, San Diego, CA.
|
Progress 07/06/15 to 09/30/15
Outputs
Target Audience:Undergraduate training: ANSC undergraduate students are exposed to the demands of research. Each participates in a portion of the project (collection, isolation, and culture of spermatogonial stem cells). Graduate student training: A doctoral student who has her own research project (isolation, culture, and characterization of feline ESC and iPSC) has been in charge of training the undergraduates student. Thus she has gained experience in mentoring. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided opportunities for graduate students (one graduated 2014 andone graduating summer 2018) and undergraduate students (usually one to three per academic year). How have the results been disseminated to communities of interest?Abstracts and manuscripts have been published on the work of Dr. Lindsey Vansandt (graduated 2015). Abstract have been published, one manuscript has been submittedand a second is in preparation for submission by Ran Zhou who will be graduating summer 2018. What do you plan to do during the next reporting period to accomplish the goals?I will continue to work with the undergraduates on the SSC project.
Impacts
What was accomplished under these goals?
1: Graduate project: Feline ESC-like cells have obtained from in vitro produced blastocyst andcultured with a variety of cytokines to identify conditions which will extend their time in culture. A combination of human LIF, feline LIF, GSK3 inhibitor,MEK inhibitor, and ROCK inhibitor was shown to extend the number of passages over which the cells maintain expression of the pluripotency transcription factors (NANOG, POU5F1, and SOX2). While time in culture was extended, these ESC-like cells have characteristics more similar to primed state stem cell than naive (ground state) stem cells. Therefore additional optimization of culture conditions is needed. Feline induced pluripotent stemcell (iPSC) have been produced using lenti-viral vectors containing tet-on humanNANOG,POU5F1,SOX2,KLF4, and CMYC transcription factors.After removal of doxycycline the transgenes are silenced but the endogenous plurpotency-related genes are expressed although not in a sufficiently balanced amount for maintenance of a naive state. The iPSC cells have characteristics more similar to primed state.As for the ESC-like cells, further optimization is needed. 2. Undergraduate Project: The methods for isolation and culture of feline spermatogonial stems cells (SSC) were established previously by a former graduate student. The undergraduates are following these procedures andhave been successful at isolationand short-term maintanance. They are working on extending the number of passages and transfecting the SSC with reporter genes. 3. Undergraduate Project: 3-D culture of spermatogonial cells (testicular tissue): Conditions which support in vitro spermatogenesis in the mouse have been shown not to work for feline testicular tissue. Small pieces of feline tissue cultured on gel stacks can only survive less than a week. Alterations in conditions are being tested.
Publications
|
|