scholarly journals Molecular species of phospholipids in a murine stem-cell line responsive to erythropoietin

1996 ◽  
Vol 314 (3) ◽  
pp. 861-867 ◽  
Author(s):  
Barbara S. BECKMAN ◽  
Conrad MALLIA ◽  
Sanda CLEJAN

The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (×6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC.

Impact ◽  
2018 ◽  
Vol 2018 (3) ◽  
pp. 26-28
Author(s):  
Jonathan Dawson ◽  
Richard Oreffo

Gels made from clay could provide an environment able to stimulate stem-cells due to their ability to bind biological molecules. That molecules stick to clay has been known by scientists since the 1960s. Doctors observed that absorption into the blood stream of certain drugs was severely reduced when patients were also receiving clay-based antacid or anti-diarrhoeal treatments. This curious phenomenon was realized to be due to binding of the drugs by clay particles. This interaction is now routinely harnessed in the design of tablets to carefully control the release and action of a drug. Dr Dawson now proposes to use this property of clay to create micro-environments that could stimulate stem cells to regenerate damaged tissues such as bone, cartilage or skin. The rich electrostatic properties of nano (1 millionth of a millimetre) -scale clay particles which mediate these interactions could allow two hurdles facing the development of stem-cell based regenerative therapies to be overcome simultaneously. The first challenge - to deliver and hold stem cells at the right location in the body - is met by the ability of clays to self-organise into gels via the electrostatic interactions of the particles with each other. Cells mixed with a low concentration (less than 4%) of clay particles can be injected into the body and held in the right place by the gel, eliminating, in many situations, the need for surgery. Clay particles can also interact with large structural molecules (polymers) which are frequently used in the development of materials (or 'scaffolds'), designed to host stem cells. These interactions can greatly improve the strength of such structures and could be applied to preserve their stability at the site of injury until regeneration is complete. While several gels and scaffold materials have been designed to deliver and hold stem cells at the site of regeneration, the ability of clay nanoparticles to overcome a second critical hurdle facing stem-cell therapy is what makes them especially exciting. Essential to directing the activity of stem-cells is the carefully controlled provision of key biological signalling molecules. However, the open structures of conventional scaffolds or gels, while essential for the diffusion of nutrients to the cells, means their ability to hold the signalling molecules in the same location as the cells is limited. The ability of clay nano-particles to bind biological molecules presents a unique opportunity to create local environments at a site of injury or disease that can stimulate and control stem-cell driven repair. Dr Dawson's early studies investigated the ability of clay gels to stimulate the growth of new blood vessels by incorporating a key molecular signal that stimulates this process, vascular endothelial growth factor (VEGF). In a manner reminiscent of the observations made in the 60s, Dr Dawson and colleagues observed that adding a drop of clay gel to a solution containing VEGF caused, after a few hours, the disappearance of VEGF from the solution as it became bound to the gel. When placed in an experimental injury model, the gel-bound VEGF stimulated a cluster of new blood vessels to form. These exciting results indicate the potential of clay nanoparticles to create tailor-made micro-environments to foster stem cell regeneration. Dr Dawson is developing this approach as a means of first exploring the biological signals necessary to successfully control stem cell behaviour for regeneration and then, using the same approach, to provide stem cells with these signals to stimulate regeneration in the body. The project will seek to test this approach to regenerate bone lost to cancer or hip replacement failure. If successful the same technology may be applied to harness stem cells for the treatment of a whole host of different scenarios, from burn victims to those suffering with diabetes or Parkinson's.


2021 ◽  
Vol 8 (12) ◽  
pp. 202
Author(s):  
Nancy M. S. Hussein ◽  
Josie L. Meade ◽  
Hemant Pandit ◽  
Elena Jones ◽  
Reem El-Gendy

Periodontitis and diabetes mellitus (DM) are two of the most common and challenging health problems worldwide and they affect each other mutually and adversely. Current periodontal therapies have unpredictable outcome in diabetic patients. Periodontal tissue engineering is a challenging but promising approach that aims at restoring periodontal tissues using one or all of the following: stem cells, signalling molecules and scaffolds. Mesenchymal stem cells (MSCs) and insulin-like growth factor (IGF) represent ideal examples of stem cells and signalling molecules. This review outlines the most recent updates in characterizing MSCs isolated from diabetics to fully understand why diabetics are more prone to periodontitis that theoretically reflect the impaired regenerative capabilities of their native stem cells. This characterisation is of utmost importance to enhance autologous stem cells based tissue regeneration in diabetic patients using both MSCs and members of IGF axis.


2012 ◽  
Vol 02 (01) ◽  
pp. 5-14 ◽  
Author(s):  
Sonal R. Tuljapurkar ◽  
John D. Jackson ◽  
Susan K. Brusnahan ◽  
Barbara J. O’Kane ◽  
John G. Sharp

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Elaheh Karbassi ◽  
Alessandro Bertero ◽  
Shin Kadota ◽  
Paul Fields ◽  
Lil Pabon ◽  
...  

Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) applications for cell therapy and disease modeling are limited due to the cells’ lack of resemblance structurally and functionally to adult cardiomyocytes. To understand hPSC-CM maturation, we characterized two established approaches to mature cardiomyocytes—long term culture (aging of cells in a dish) and in vivo transplantation to an infarcted adult rat heart. RNA sequencing of hPSC-CMs from these systems demonstrated that in vivo transplantation is much more effective in maturing hPSC-CMs, inducing a more adult-like cardiac gene program (e.g. upregulation of TNNI3, MYL2, SCN5A ), compared to cells kept in culture up to one year. Using this dataset, we identified candidate drivers of hPSC-CM maturation, including transcription factors and chromatin regulators, that we hypothesize are necessary to program hPSC-CMs to an adult-like state. To test the relationship between transcription factor regulation and hPSC-CM maturation, we developed a constitutive CRISPR activation (CRISPRa) pluripotent stem cell line to upregulate these transcriptional regulators upon addition of guide RNAs (gRNA). This cell line expresses nuclease-deficient Cas9 fused to the transcriptional activator VPR (dCas9-VPR), driven by the strong CAG promoter and targeted to the AAVS1 safe harbor site. In pluripotent stem cells, target genes are upregulated up to 150-fold when gRNA is present; however, after differentiation into cardiomyocytes, dCas9-VPR transgene expression is silenced, and dCas9-VPR levels are insufficient to activate gRNA-targeted genes. To optimize CRISPRa for cardiomyocyte applications, we are generating alternative stem cell lines with dCas9-VPR targeted to the human ROSA26 safe harbor site or driven by a cardiac-specific troponin T promoter, testing the regulation of transgene expression mediated by safe harbor site or promoter respectively. The characterization of these CRISPRa cell lines provides insights into CRISPR expression regulation and genome engineering strategies for applications in stem cells and hPSC-CMs. We will use this system to screen for maturation regulators and identify key combinations that are effective in programming hPSC-CMs towards an adult-like state.


2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
G. Forte ◽  
O. Franzese ◽  
S. Pagliari ◽  
F. Pagliari ◽  
A. M. Di Francesco ◽  
...  

An immortalized murine mesenchymal stem cell line (mTERT-MSC) enriched forLinneg/Sca-1posfraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 μm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.


2020 ◽  
Author(s):  
Blair W. Benham-Pyle ◽  
Carolyn E. Brewster ◽  
Aubrey M. Kent ◽  
Frederick G. Mann ◽  
Shiyuan Chen ◽  
...  

AbstractRegeneration requires functional coordination of stem cells, their progeny, and differentiated cells. Past studies have focused on regulation of stem cell identity and proliferation near to the wound-site, but less is known about contributions made by differentiated cells distant to the injury. Here, we present a comprehensive atlas of whole-body regeneration over time and identify rare, transient, somatic cell states induced by injury and required for regeneration. To characterize amputation-specific signaling across a whole animal, 299,998 single-cell transcriptomes were captured from planarian tissue fragments competent and incompetent to regenerate. Amputation-specific cell states were rare, non-uniformly distributed across tissues, and particularly enriched in muscle (mesoderm), epidermis (ectoderm), and intestine (endoderm). Moreover, RNAi-mediated knockdown of genes up-regulated in amputation-specific cell states drastically reduced regenerative capacity. These results identify novel cell states and molecules required for whole-body regeneration and indicate that regenerative capacity requires transcriptional plasticity in a rare subset of differentiated cells.


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