Parthenogenesis in mammals: pros and cons in pluripotent cell derivation

2011 ◽  
Vol 6 (5) ◽  
pp. 770-775
Author(s):  
Georgia Pennarossa ◽  
Alessio Paffoni ◽  
Guido Ragni ◽  
Fulvio Gandolfi ◽  
Tiziana Brevini
Keyword(s):  
Cell Biology ◽  
Ad Hoc ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Embryos ◽  
In Vitro Production ◽  
Alternative Source ◽  
Depth Analysis ◽  
Mature Cell Type

AbstractEmbryonic stem cells (ESCs) represent a useful tool for cell therapy studies, however the use of embryos for their derivation give rise to ethical, religious and legal problems when applied to the human. During the last years parthenogenesis has been proposed as an alternative source to obtain ESCs. Based on the fact that parthenotes avoid many concerns surrounding the “ad hoc” in vitro production and following destruction of viable human embryos. Unfortunately many aspects related to parthenogenetic cell biology are not fully understood and still need to be elucidated. In this review we describe advantages and limits of these cells. We discuss their typical ESC morphology and high telomerase activity, which disappears after differentiation. We examine the pluripotency signature that they share with bi-parental ESCs. We review their high differentiation plasticity that allow for the derivation of several mature cell type populations when we expose these cells to adequate conditions. On the other hand, in-depth analysis demonstrated chromosome mal-segregation and altered mechanisms controlling centriole arrangement and mitotic spindle formation in these cells. We hypothesize their monoparental origin as one of the possible cause of these anomalies and suggest a great caution if a therapeutic use is considered.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ping Zhou ◽  
Jia-Min Shi ◽  
Jing-E Song ◽  
Yu Han ◽  
Hong-Jiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Pluripotent Stem Cells ◽  
Cell Counting ◽  
Cell Type ◽  
Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

scholarly journals Stimulatory Role of Glycated Fetal Bovine Serum Along with Iron on In Vitro Production of Insulin by Differentiated Mouse Embryonic Stem Cells

2011 ◽  
Vol 57 (4) ◽  
pp. 356-361
Author(s):  
Ikuo Nishigaki ◽  
Gowri Rangasamy Gunassekaran ◽  
Panjan Nagappan Venkatesan ◽  
Mandupal Chaco Sabu ◽  
Sabu Priya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Embryonic Stem ◽  
In Vitro Production ◽  
Fetal Bovine ◽  
Vitro Production

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2339-2348 ◽  
Author(s):  
B. Pain ◽  
M.E. Clark ◽  
M. Shen ◽  
H. Nakazawa ◽  
M. Sakurai ◽  
...  
Keyword(s):  
Culture System ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Telomerase Activity ◽  
Specific Antibodies ◽  
Typical Morphology ◽  
Germinal Tissue

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Epigenetic features in regulation of telomeres and telomerase in stem cells

2021 ◽  
Author(s):  
Fatma Dogan ◽  
Nicholas R. Forsyth
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Cell Biology ◽  
Replicative Senescence ◽  
Telomerase Activity ◽  
Cell Phenotype ◽  
Tissue Degeneration ◽  
Unique System ◽  
Telomerase Regulation

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.

166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. K. Singh ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Tumor Rejection ◽  
Cell Surface Expression ◽  
Human Embryos ◽  
Strong Expression

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.

scholarly journals Towards structural characterization of H. polymorpha telomerase components

2014 ◽  
Vol 70 (a1) ◽  
pp. C816-C816
Author(s):  
Johanna Kallio ◽  
Elena Rodina ◽  
Maria Zvereva ◽  
Olga Petrova ◽  
Alexandr Malyavko ◽  
...  
Keyword(s):  
Cell Division ◽  
Structural Information ◽  
Hansenula Polymorpha ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Premature Aging ◽  
Telomeric Dna ◽  
Terminal Domain ◽  
Protein Components

Telomeres are regions of non-coding DNA that cap the chromosomes, preventing the loss of coding DNA during cell division and contributing to chromosomal stability. In actively dividing cells, such as embryonic stem cells, the telomeres need to elongated by telomerase. The telomerase complex consist of the enzyme telomerase reverse transcriptase (TERT), telomerase RNA (TR) and additional proteins. TERT and TR are required for the telomerase activity in vitro. Telomerase is active in vast majority of the cancer cells ensuring continuous cell division and tumor growth. Syndromes leading to premature aging are often associated with short telomeres. Finding ways to regulate the telomerase activity would help to advance therapies for these conditions. However, the structural information available of the telomerase complex is very limited. We have chosen thermophilic yeast Hansenula polymorpha as a model system due to the stability of its proteins. The N-terminal domain of the TERT is essential for telomerase activity and possibly is involved in binding of TR, telomeric DNA and additional protein components of the telomerase complex. We have crystallised the N-terminal domain of H. polymorpha TERT and, in lack of a homologious structure, produced a seleno-methionine derivative of the protein. MAD data on N-terminal domain has been collected to resolution of 2.0 Å at the PETRA-III beamline P13 (EMBL/DESY) in Hamburg. We will discuss the structure-function relationship of the N-domain and the whole TERT component.

scholarly journals Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery

Blood ◽  
2011 ◽  
Vol 118 (12) ◽  
pp. 3254-3262 ◽  
Author(s):  
Massimo Giuliani ◽  
Noufissa Oudrhiri ◽  
Zaeem M. Noman ◽  
Amelia Vernochet ◽  
Salem Chouaib ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nk Cell ◽  
Embryonic Stem ◽  
Attractive Alternative ◽  
Target Cells ◽  
Alternative Source ◽  
Activation Markers ◽  
Induced Pluripotent

Abstract A major issue in immunosuppressive biotherapy is the use of mesenchymal stem cells (MSCs) that harbor regulatory capacity. However, currently used bone marrow-derived MSCs (BM-MSCs) are short-lived and cannot assure long lasting immunoregulatory function both in vitro and in vivo. Consequently, we have generated MSCs from human induced pluripotent stem (IPS-MSCs) cells that share similar properties with embryonic stem cells (ES-MSCs). Herein, we compared the immunoregulatory properties of ES/IPS-MSCs with those of BM-MSCs and showed, for the first time, that IPS-derived MSCs display remarkable inhibition of NK-cell proliferation and cytolytic function in a similar way to ES-MSCs. Both MSCs disrupt NK-cell cytolytic machinery in the same fashion that BM-MSCs, by down-regulating the expression of different activation markers and ERK1/2 signaling, leading to an impairment to form immunologic synapses with target cells and, therefore, secretion of cytotoxic granules. In addition, they are more resistant than adult BM-MSCs to preactivated NK cells. IPS-MSCs could represent an attractive alternative source of immunoregulatory cells, and their capacity to impair NK-cell cytotoxicity constitutes a complex mechanism to prevent allograft rejection.

Hepatic differentiation of pluripotent stem cells

2009 ◽  
Vol 390 (10) ◽  
Author(s):  
Komal Loya ◽  
Reto Eggenschwiler ◽  
Kinarm Ko ◽  
Malte Sgodda ◽  
Francoise André ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Pluripotent Stem Cells ◽  
Ethical Issues ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hepatic Differentiation ◽  
Alternative Source ◽  
Organ Specific

Abstract In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

Cardiomyocyte culture — an update on the in vitro cardiovascular model and future challenges

2013 ◽  
Vol 91 (12) ◽  
pp. 985-998 ◽  
Author(s):  
Sreejit Parameswaran ◽  
Sujeet Kumar ◽  
Rama Shanker Verma ◽  
Rajendra K. Sharma
Keyword(s):  
Stem Cells ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Small Animal ◽  
Heart Tissue ◽  
Cellular Level ◽  
Future Challenges ◽  
Vitro Model ◽  
Induced Pluripotent

The success of any work with isolated cardiomyocytes depends on the reproducibility of cell isolation, because the cells do not divide. To date, there is no suitable in vitro model to study human adult cardiac cell biology. Although embryonic stem cells and induced pluripotent stem cells are able to differentiate into cardiomyocytes in vitro, the efficiency of this process is low. Isolation and expansion of human cardiomyocyte progenitor cells from cardiac surgical waste or, alternatively, from fetal heart tissue is another option. However, to overcome various issues related to human tissue usage, especially ethical concerns, researchers use large- and small-animal models to study cardiac pathophysiology. A simple model to study the changes at the cellular level is cultures of cardiomyocytes. Although primary murine cardiomyocyte cultures have their own advantages and drawbacks, alternative strategies have been developed in the last two decades to minimise animal usage and interspecies differences. This review discusses the use of freshly isolated murine cardiomyocytes and cardiomyocyte alternatives for use in cardiac disease models and other related studies.

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