The Efficiency of Cell Fusion-Based Reprogramming Is Affected by the Somatic Cell Type and the In Vitro Age of Somatic Cells

Methods for directly turning a somatic cell type into another type (a process referred to as transdifferentiation) would be beneficial for producing replacement cells for therapeutic applications. Adult stem cells have been shown to display a broader differentiation potential than anticipated and may contribute to tissues other than those in which they reside. In addition, novel transdifferentiation strategies are being developed. I report recent results on the functional reprogramming of a somatic cell using a nuclear and cytoplasmic extract derived from another somatic cell type. The reprogramming of 293T fibroblasts in an extract from T cells is evidenced by nuclear uptake and the assembly of transcription factors, induction of activity of a chromatin remodelling complex, changes in chromatin composition and activation of lymphoid cell–specific genes. The reprogrammed cells express T–cell–specific surface molecules and a complex regulatory function. Reprogramming cells in cell–free extracts may create possibilities for producing replacement cells for therapeutic applications. The system may also constitute a powerful tool to examine the mechanisms of nuclear reprogramming, at least as they occur in vitro .

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GC-1 spg cells are most similar to Leydig cells, a testis somatic cell-type, and not germ cells

10.1101/2021.01.07.425754 ◽

2021 ◽

Author(s):

Andrew R Norman ◽

Lauren Byrnes ◽

Jeremy R Reiter

Keyword(s):

Germ Cell ◽

Somatic Cell ◽

Cell Line ◽

Germ Cells ◽

Leydig Cells ◽

Adult Mouse ◽

Somatic Cells ◽

Cell Type ◽

Immortalized Cell ◽

Somatic Cell Type

GC-1 spg is an immortalized cell line derived from an adult mouse testis and reported to be most similar to spermatocytes, a male germ cell-type. However, immunofluorescence indicates that GC-1 spg cells express WT1, a marker of testis somatic cells, and do not express markers of germ cells. Transcriptomic profiling indicate GC-1 cells are most similar to Leydig cells. Therefore, we conclude that GC-1 spg cells are most similar to testis somatic cells.

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The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

Viruses ◽

10.3390/v12060629 ◽

2020 ◽

Vol 12 (6) ◽

pp. 629 ◽

Cited By ~ 36

Author(s):

Mizuki Yamamoto ◽

Maki Kiso ◽

Yuko Sakai-Tagawa ◽

Kiyoko Iwatsuki-Horimoto ◽

Masaki Imai ◽

...

Keyword(s):

Cell Fusion ◽

Assay System ◽

Dependent Manner ◽

Cell Type ◽

S Protein ◽

Candidate Drug ◽

A Cell ◽

Transmembrane Serine Protease ◽

Nafamostat Mesylate

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 μM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat’s safety, make it a likely candidate drug to treat COVID-19.

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65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab65 ◽

2008 ◽

Vol 20 (1) ◽

pp. 113

Author(s):

H. M. Zhou ◽

B. S. Li ◽

L. J. Zhang

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Calf Serum ◽

Cumulus Cells ◽

Developmental Competence ◽

Developmental Potential ◽

Reconstructed Embryos ◽

Electrical Pulses ◽

Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

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Effect of somatic cell type on bovine embryonic development in co-culture

Theriogenology ◽

10.1016/0093-691x(91)90245-9 ◽

1991 ◽

Vol 35 (1) ◽

pp. 269 ◽

Cited By ~ 6

Author(s):

J.M. Scodras ◽

J.W. Pollard ◽

K.J. Betteridge

Keyword(s):

Embryonic Development ◽

Somatic Cell ◽

Cell Type ◽

Somatic Cell Type

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Fusion as the result of sperm–somatic cell interaction

Reproduction ◽

10.1530/rep-08-0316 ◽

2009 ◽

Vol 138 (4) ◽

pp. 679-687 ◽

Cited By ~ 9

Author(s):

M Mattioli ◽

A Gloria ◽

A Mauro ◽

L Gioia ◽

B Barboni

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Cell Types ◽

Cell Interaction ◽

Fluorescent Dye ◽

Cell Plasma ◽

Molecular Machinery ◽

Different Cell Types ◽

Boar Spermatozoa

The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm–egg fusion. Boar spermatozoa were capacitatedin vitroand then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin α6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.

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31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab31 ◽

2006 ◽

Vol 18 (2) ◽

pp. 124 ◽

Cited By ~ 1

Author(s):

F. Du ◽

J. Xu ◽

S. Gao ◽

L. Y. Sung ◽

D. Stone ◽

...

Keyword(s):

Somatic Cell ◽

Cell Fusion ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Cumulus Cells ◽

Full Term ◽

Fusion Method ◽

Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

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Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

Current Gene Therapy ◽

10.2174/1566523219666190902154511 ◽

2019 ◽

Vol 19 (4) ◽

pp. 248-254

Author(s):

Marianne Lehmann ◽

Martina Canatelli-Mallat ◽

Priscila Chiavellini ◽

Gloria M. Cónsole ◽

Maria D. Gallardo ◽

...

Keyword(s):

Stem Cells ◽

Somatic Cell ◽

Fluorescent Protein ◽

Somatic Cells ◽

Cell Types ◽

Cell Reprogramming ◽

Green Fluorescent ◽

Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

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TNFα-mediated plasminogen activation on neutrophils is involved in the high plasmin activity in mammary secretion of drying-off cows

Journal of Dairy Research ◽

10.1017/s0022029909990069 ◽

2009 ◽

Vol 76 (4) ◽

pp. 459-468 ◽

Cited By ~ 6

Author(s):

Wen K Chou ◽

Ting C Yu ◽

Shuen E Chen ◽

Ho C Peh ◽

Wen B Liu ◽

...

Keyword(s):

Somatic Cell ◽

Specific Activity ◽

Somatic Cells ◽

Polymorphonuclear Neutrophil ◽

Bovine Blood ◽

Plasmin Activity ◽

Vitro Assay ◽

Cell Counts ◽

Tnf Α

Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-α in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (Pm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-α antibody, and was further up-regulated by exogenous bovine TNF-α. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-α decreased immediately following milk stasis. Iimmunoblotting revealed that although both 26-kDa pro-TNF-α and 17-kDa TNF-α were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-α was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-α-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-α on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-α level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary gland.

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The Stability of the Induced Epigenetic Programs

Comparative and Functional Genomics ◽

10.1155/2012/434529 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Maria J. Barrero

Keyword(s):

Stem Cells ◽

Somatic Cells ◽

Cell Types ◽

Specific Cell ◽

Cell Type ◽

Pluripotent Cells ◽

Potential Applications ◽

The Stability

For many years scientists have been attracted to the possibility of changing cell identity. In the last decades seminal discoveries have shown that it is possible to reprogram somatic cells into pluripotent cells and even to transdifferentiate one cell type into another. In view of the potential applications that generating specific cell types in the laboratory can offer for cell-based therapies, the next important questions relate to the quality of the induced cell types. Importantly, epigenetic aberrations in reprogrammed cells have been correlated with defects in differentiation. Therefore, a look at the epigenome and understanding how different regulators can shape it appear fundamental to anticipate potential therapeutic pitfalls. This paper covers these epigenetic aspects in stem cells, differentiation, and reprogramming and discusses their importance for the safety of in vitro engineered cell types.

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