scholarly journals Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Sujeong Jang ◽  
Jong-Seong Park ◽  
Han-Seong Jeong
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Differentiation ◽  
Neural Induction ◽  
Stem Cell Differentiation ◽  
Human Adipose Tissue ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Jnk Pathway ◽  
Expression Levels

Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

Oct4 and Its Pseudogenes Confuse Stem Cell Research.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3700-3700
Author(s):  
Stefanie Liedtke ◽  
Jürgen Enczmann ◽  
Simon Waclawczyk ◽  
Peter Wernet ◽  
Gesine Kögler
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Differentiated Cells ◽  
Alternatively Spliced

Abstract Octamer-binding transcription factor 4 (Oct4) encodes a nuclear protein that belongs to a family of transcription factors containing the POU DNA binding domain. It is specifically expressed in embryonic stem cells but can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. The expression of Oct4 is down-regulated coincident with stem cell differentiation and loss of expression leading to differentiation. It plays a critical role for maintaining pluripotency and self-renewal of embryonic stem cells. However, the usefulness of Oct4 as a pluripotency marker was challenged recently. More and more data seem to support that Oct4 is expressed on a variety of differentiated cells, including peripheral blood mononuclear cells. Taking into account that RT-PCR can potentially generate experimental artifacts due to pseudogene transcripts, the existence of Oct4 pseudogenes should be investigated further here. Suo et al. were able to detect transcription of some Oct4 pseudogenes in cancer cell lines as well as cancer tissues. These results show that some of the known Oct4 pseudogenes are transcribed in vivo and therefore could lead to RT-PCR artifacts. However this known problem was not seriously taken into consideration in recent publications on adult stem cells and tissue analysis referring to Oct4. We started with an initial alignment of Oct4 compared to its alternative splice variants as well as its pseudogenes. This alignment served as a prerequisite for an exact primer design. First the sequence and organization of the functional human Oct4 gene were clarified to allow comparison to the pseudogenes and alternatively spliced transcripts. The NCBI human EST database was searched and the UniGene cluster for Oct4 (NM_002701) examined. This yielded 13 mRNA sequences and 129 EST sequences. An additional BLASTn search of the human genome using single exons of Oct4 revealed several other highly similar sequences. All these hits encoded complete or partial Oct4 sequences and could therefore represent either functional members of an Oct4 gene family or pseudogenes. The fact that so many homologous sequences resemble the original Oct4 transcript makes an RT-PCR analysis difficult, because a lot of artifacts can arise during amplification. Therefore primers were designed which are able to exclude amplification of all unwanted transcripts. To conclude, based on the fact that the expression of Oct4 has been reported in adult stem cells as well as in a variety of differentiated cells the possibility cannot be excluded that the detected Oct4 signal came from alternatively spliced or Oct4 pseudogene transcripts. As shown here, an exact design of Oct4-specific primers is an inevitable prerequisite for appropriate RT-PCR analysis. In addition, a careful comparison of quantitative differences to human embryonic stem cells should be present too, before cells are described as embryonic like cells. We hope that our findings will help other stem cell researchers to find their appropriate tools especially for RT-PCR analysis and give an example how later problematic artifacts can be ruled out from the beginning by a detailed alignment as a prerequisite for designing appropriate primers.

scholarly journals Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2154-2161 ◽  
Author(s):  
AG Turhan ◽  
FM Lemoine ◽  
C Debert ◽  
ML Bonnet ◽  
C Baillou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Promyelocytic Leukemia ◽  
Cell Sorting ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Pcr Analysis ◽  
Cell Populations ◽  
Rt Pcr ◽  
Hematopoietic Stem

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38-subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte- macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.

scholarly journals Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. Results ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

scholarly journals Successful Transplantation of Spermatogonial Stem Cells Into the Seminiferous Tubules of Busulfan-treated Mice

2020 ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology - immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer.Results: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P< 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

scholarly journals DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells

2021 ◽  
Vol 22 (8) ◽  
pp. 4011
Author(s):  
Brianna Chen ◽  
Dylan McCuaig-Walton ◽  
Sean Tan ◽  
Andrew P. Montgomery ◽  
Bryan W. Day ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Critical Role ◽  
Stem Cell Differentiation ◽  
Neural Progenitor Cell ◽  
Cellular Heterogeneity ◽  
Differentiation Potential ◽  
Glioblastoma Stem Cells ◽  
Glioblastoma Stem Cell

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

scholarly journals Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

2015 ◽  
Vol 35 (10) ◽  
pp. 1700-1711 ◽  
Author(s):  
Fenfang Chen ◽  
Xia Lin ◽  
Pinglong Xu ◽  
Zhengmao Zhang ◽  
Yanzhen Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Mesenchymal Stem Cell ◽  
Nuclear Export ◽  
Stem Cell Differentiation ◽  
Bmp Signaling ◽  
Dependent Manner ◽  
Mesenchymal Stem Cell Differentiation

Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation.

Magnetic field assisted stem cell differentiation – role of substrate magnetization in osteogenesis

2015 ◽  
Vol 3 (16) ◽  
pp. 3150-3168 ◽  
Author(s):  
Sunil Kumar Boda ◽  
Greeshma Thrivikraman ◽  
Bikramjit Basu
Keyword(s):  
Magnetic Field ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Bone Tissue Engineering ◽  
Human Mesenchymal Stem Cells ◽  
Stem Cell Differentiation

Substrate magnetization as a tool for modulating the osteogenesis of human mesenchymal stem cells for bone tissue engineering applications.

scholarly journals Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

2012 ◽  
Vol 287 (44) ◽  
pp. 36777-36791 ◽  
Author(s):  
Hiroaki Fujimori ◽  
Mima Shikanai ◽  
Hirobumi Teraoka ◽  
Mitsuko Masutani ◽  
Ken-ichi Yoshioka
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation

Biomaterials Nano Geometry for Control of Stem Cell Differentiation

2009 ◽  
Vol 1239 ◽  
Author(s):  
Karla Brammer ◽  
Seunghan Oh ◽  
Sungho Jin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Stem Cell Differentiation ◽  
Human Mesenchymal Stem Cell ◽  
Oxide Surface ◽  
Specific Cell ◽  
Implant Surface ◽  
Multipotent Stem Cells

AbstractTwo important goals in stem cell research are to control the cell proliferation without differentiation, and also to direct the differentiation into a specific cell lineage when desired. Recent studies indicate that the nanostructures substantially influence the stem cell behavior. It is well known that mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into stromal lineages such as adipocyte, chondrocyte, fibroblast, myocyte, and osteoblast cell types. By examining the cellular behavior of MSCs cultured in vitro on nanostructures, some understanding of the effects that the nanostructures have on the stem cell’s response has been obtained. Here we demonstrate that TiO2 nanotubes produced by anodization on Ti implant surface can regulate human mesenchymal stem cell (hMSC) differentiation towards an osteoblast lineage in the absence of osteogenic inducing factors. Altering the dimensions of nanotubular-shaped titanium oxide surface structures independently allowed either augmented human mesenchymal stem cell (hMSC) adhesion at smaller diameter levels or a specific differentiation of hMSCs into osteoblasts using only the geometric cues. Small (˜30 nm diameter) nanotubes promoted adhesion without noticeable differentiation, while larger (˜70 - 100 nm diameter) nanotubes elicited a dramatic, ˜10 fold stem cell elongation, which induced cytoskeletal stress and selective differentiation into osteoblast-like cells, offering a promising nanotechnology-based route for novel orthopaedics-related hMSC treatments. The fact that a guided and preferential osteogenic differentiation of stem cells can be achieved using substrate nanotopography alone without using potentially toxic, differentiation-inducing chemical agents is significant, which can be useful for future development of novel and enhanced stem cell control and therapeutic implant development.

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