scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

2020 ◽  
Vol 8 (4) ◽  
pp. 139-145
Author(s):  
Rut Bryl ◽  
Claudia Dompe ◽  
Maurycy Jankowski ◽  
Katarzyna Stefańska ◽  
Afsaneh Golkar Narenji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Stem Cell Marker ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Long Term Culture ◽  
Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

scholarly journals Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 560-567 ◽  
Author(s):  
David G. Kent ◽  
Brad J. Dykstra ◽  
Jay Cheyne ◽  
Elaine Ma ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Biologic Function

Abstract Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division.

scholarly journals Etoposide-Initiated MLL Rearrangements Detected at High Frequency in Human Primitive Hematopoietic Stem Cells with In Vitro and In Vivo Long-Term Repopulating Potential.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 98-98 ◽  
Author(s):  
Jolanta Libura ◽  
Marueen Ward ◽  
Grzegorz Przybylski ◽  
Christine Richardson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
P Value ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Rearrangements involving the MLL gene locus at chromosome band 11q23 are observed in therapy-related acute myeloid leukemia and myelodysplastic syndromes following treatment with topoisomerase II (topoII) inhibitors including etoposide. We have shown that one hour of etoposide exposure (20–50 μM) stimulates stable MLL rearrangements in primary human CD34+ cells and that the spectrum of repair products within MLL gene is broader than so far described (Libura et al, Blood, 2005). Clinical data suggest that MLL-associated malignant leukemias originate within primitive hematopietic stem cells capable of differentiation into all hematopoietic lineages and repopulation of myelo-ablated hosts. These cells can be analyzed using the in vivo NOD-SCID mouse model as well as the in vitro long-term culture initiating cell (LTC-IC) assay. We adopted our in vitro CD34+ cell culture model to investigate the impact of etoposide exposure on the most primitive hematopoietic stem cells using parallel assays for LTC-IC and NOD-SCID Repopulating Cells (SRC). Following etoposide exposure (20–50 μM for 1 hour), and 48–96 hours recovery in vitro, untreated control and etoposide-treated CD34+ cells were either seeded in LTC-IC with a supportive feeder layer (Stem Cell Technologies, Inc.), or injected into NOD-SCID mice (0.1–1.5x106 cells per mouse). After 12 weeks, both LTC-IC cultures and bone marrow cells from NOD-SCID mice were seeded in methylcellulose media supplemented with growth factors that promote only human cell colony formation. An increased number of colonies in etoposide-treated samples was obtained from LTC-IC cultures in 3 out of 5 experiments (p value&lt;0.05). This increase in colony number was more dramatic in etoposide-treated samples from NOD-SCID bone marrow (57 versus 0, 8 versus 0). These data demonstrate that etoposide exposure can significantly alter the potential of early hematopoietic stem cells to survive and proliferate both in vitro and in vivo. Injection of as few as 3x105 CD34+ cells into a NOD-SCID mouse was sufficient to obtain methylcellulose colonies, suggesting that this method can be used for the analysis of cells obtained from a single patient sample. Mutation analysis of human methylcellulose colonies derived from both LTC-IC and NOD-SCID was performed by inverse PCR and ligation-mediated PCR followed by sequencing. This analysis revealed that rearrangements originating within the MLL breakpoint cluster region (bcr) were present in 12 out of 29 colonies from etoposide-treated samples versus 5 out of 39 colonies from control samples (p value &lt;0.01), demonstrating that etoposide exposure promotes stable rearrangements within a hematopoietic stem cell compartment with significant proliferative potential. Eight of the 17 events were sequenced, and showed 6 MLL tandem duplications within intron 8, one complex translocation between MLL and chr.15 and tandem duplication, and one event with foreign sequence of unknown origin. Our data are the first report of the spectrum and frequency of MLL rearrangements following topo II inhibitor exposure in a cell population thought to be the target for recombinogenic events leading to therapy-related leukemias.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Lihua Yin ◽  
Wenxiao Cheng ◽  
Zishun Qin ◽  
Hongdou Yu ◽  
Zhanhai Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Trabecular Structure ◽  
Control Group ◽  
Periodontal Ligament Stem Cells ◽  
Expression Levels ◽  
Proliferation And Differentiation

This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs) induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2,COL1A2, OPN, and OCN) had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

scholarly journals Tenuigenin promotes the osteogenic differentiation of bone mesenchymal stem cells in vitro and in vivo

2016 ◽  
Vol 367 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Hua-ji Jiang ◽  
Xing-gui Tian ◽  
Shou-bin Huang ◽  
Guo-rong Chen ◽  
Min-jun Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bone Mesenchymal Stem Cells

scholarly journals A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Endocrine ◽  
2021 ◽  
Author(s):  
Miriam E. A. Tschaffon ◽  
Stefan O. Reber ◽  
Astrid Schoppa ◽  
Sayantan Nandi ◽  
Ion C. Cirstea ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
In Vitro Assay ◽  
Immunofluorescent Staining ◽  
Marker Genes ◽  
Osteogenic Differentiation Medium ◽  
Vitro Assay ◽  
Osteogenic Cells ◽  
Osteogenic Lineage

Abstract Purpose Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage. Methods Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell–matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium. Results Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage. Conclusion We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

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