scholarly journals Designing Microfluidic Devices to Sort Haematopoietic Stem Cells Based on Their Mechanical Properties

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Mingming Du ◽  
Dean Kavanagh ◽  
Zhibing Zhang ◽  
Neena Kalia
Keyword(s):  
Mechanical Properties ◽  
Stem Cells ◽  
Cellular Therapy ◽  
Microfluidic Devices ◽  
High Flow ◽  
Cell Diameter ◽  
Label Free ◽  
Haematopoietic Stem Cells ◽  
Flow Rates

Aim. Few haematopoietic stem cells (HSCs) injected systemically for therapeutic purposes actually reach sites of injury as the vast majority become entrapped within pulmonary capillaries. One promising approach to maintain circulating HSC numbers would be to separate subpopulations with smaller size and/or greater deformability from a heterogeneous population. This study tested whether this could be achieved using label-free microfluidic devices.Methods. 2 straight (A-B) and 3 spiral (C-E) devices were fabricated with different dimensions. Cell sorting was performed at different flow rates after which cell diameter and stiffness were determined using micromanipulation. Cells isolated using the most efficient device were tested intravitally for their ability to home to the mouse injured gut.Results. Only straight Device B at a high flow rate separated HSCs with different mechanical properties. Side outlets collected mostly deformable cells (nominal rupture stress/σR=6.81 kPa; coefficient of variation/CV=0.31) at a throughput of2.3×105cells/min. All spiral devices at high flow rates separated HSCs with different stiffness and size. Inner outlets collected mostly deformable cells in Devices C (σR=25.06 kPa;CV=0.26), D (σR=22.21 kPa;CV=0.41), and E (σR=29.26 kPa;CV=0.27) at throughputs of2.3×105cells/min,1.5×105cells/min, and1.6×105cells/min, respectively. Since Device C separated cells with higher efficiency and throughput, it was utilized to test the homing ability of separated cellsin vivo. Significantly more deformable cells were observed trafficking through the injured gut—interestingly, increased retention was not observed.Conclusion. This study applied microfluidics to separate subpopulations from one stem cell type based on their intrinsic mechanical heterogeneity. Fluid dynamics within curved devices most effectively separated HSCs. Such devices may benefit cellular therapy.

P5379Modification with CREKA improves cell retention of myocardial ischemia reperfusion

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Chen ◽  
Y N Song ◽  
Z Y Huang
Keyword(s):  
Stem Cells ◽  
Functional Recovery ◽  
Cellular Therapy ◽  
Tissue Injury ◽  
Ischemia Reperfusion ◽  
High Specificity ◽  
Homing Peptide ◽  
Structural Preservation

Abstract Background Poor cell homing limits efficacy of cardiac cellular therapy. The cysteine–arginine–glutamic acid–lysine–alanine (CREKA) homing peptide binds with high specificity to fibrin which is involved in repair of tissue injury. Purpose We assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. Methods CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Results Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots. CREKA-MSCs showed much more higher accumulation than unmodified MSCs in injured rat myocardium, colocalizing with fibrin and resulting in better cardiac function. Stem cell-CREKA-fibrin targeting system Conclusions Modification of MSCs with the homing peptide CREKA favored their migration and retention in the infarcted area, resulting in better structural preservation and functional recovery. Fibrin is therefore a novel target for enhancing homing of transplanted cells to injured myocardium and the fibrin-targeting delivery system represents a generalizable platform technology for regenerative medicine.

scholarly journals In vivo label-free measurement of lymph flow velocity and volumetric flow rates using Doppler optical coherence tomography

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Cedric Blatter ◽  
Eelco F. J. Meijer ◽  
Ahhyun S. Nam ◽  
Dennis Jones ◽  
Brett E. Bouma ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Flow Velocity ◽  
Lymph Flow ◽  
Optical Coherence ◽  
Label Free ◽  
Flow Rates ◽  
Doppler Optical Coherence Tomography

Transcriptional Regulation of the LMO2 T-Cell Leukaemia Oncogene.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2728-2728
Author(s):  
Josette-Renée Landry ◽  
Sarah Kinston ◽  
Kathy Knezevic ◽  
Anthony R. Green ◽  
Berthold Göttgens
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Cell Leukaemia ◽  
Haematopoietic Stem Cells ◽  
Haematopoietic Cells

Abstract Transcriptional control has long been identified as a key mechanism regulating the formation and subsequent behaviour of haematopoietic stem cells. We have used a comparative genomics approach to identify transcriptional regulatory elements of the LMO2 gene, a transcriptional cofactor originally identified through its involvement in T-cell leukaemia and subsequently shown to be critical for the formation of haematopoietic stem cells and endothelial development. An initial stringent search for homology between evolutionary distant species demonstrated that, apart from the coding exons, high level of identity between mammalian, amphibian and fish sequences was restricted to the proximal promoter region of LMO2. Real-time RT-PCR expression analysis identified this promoter as the predominant source of transcription in haematopoietic tissue. Transient and stable transfections indicated that the proximal promoter was active in haematopoietic progenitor and endothelial cell lines and this activity was shown to depend on three conserved Ets sites which were bound in vivo by Elf1, Fli1 and Ets1. Transgenic analysis demonstrated that the LMO2 proximal promoter was sufficient for expression in endothelial cells in vivo. However, no haematopoietic expression was observed indicating that additional enhancers are required to mediate transcription from the proximal promoter in haematopoietic cells. To identify additional elements involved in haematopoietic expression of LMO2, we have performed a less restrictive search for conserved sequences by comparing the human, dog, rat and mouse LMO2 loci to the marsupial opossum LMO2 locus. The addition of the opossum locus, and removal of the more distant fish and amphibian sequences from the alignment, resulted in the discovery of eleven conserved regions. These sequences represent candidate haematopoietic regulatory regions as they contain conserved transcription factor binding sites (E boxes, Ets and Gata sites) previously shown to regulate several other haematopoietic genes. We will present results from a systematic analysis of these regions for enhancer activity in both haematopoietic cell lines and transgenic mice, which suggest that several of these elements indeed act as enhancers. Taken together, our experiments will provide a framework for the transcriptional hierarchies within which LMO2 exerts its function in normal haematopoietic cells. Moreover, the current studies will serve as a platform to examine potential molecular mechanisms that can cause ectopic expression of LMO2 in T-cell progenitors with the ultimate consequence of developing T-ALL.

Identification of novel circulating human embryonic blood stem cells

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1740-1747 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei Wu ◽  
Francis Karanu ◽  
Fraser Fellows ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cellular Therapy ◽  
Fetal Liver ◽  
In Utero ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Fetal Stem Cells

Abstract Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 × 105, illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized–peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies.

PTX3 secreted by human adipose-derived stem cells promotes dopaminergic neuron repair in Parkinson's disease via inhibiting apoptosis

2021 ◽  
Author(s):  
Changlin Lian ◽  
Qiongzhen Huang ◽  
Xiangyang Zhong ◽  
Zhenyan He ◽  
Boyang Liu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Stem Cells ◽  
Parkinson's Disease ◽  
Brain Slices ◽  
Human Mesenchymal Stem Cells ◽  
Substantia Nigra Pars Compacta ◽  
Therapeutic Effects ◽  
Label Free

Abstract Background Adipose-derived human mesenchymal stem cells (hADSCs) transplantation has recently emerged as a promising method in the treatment of Parkinson's disease (PD), however, the mechanism underlying has not been fully illustrated. Methods In this study, the therapeutic effects of the striatum stereotaxic injected hADSCs in 6-OHDA-induced mouse model were evaluated. Furthermore, an in vitro model of PD was constructed using tissue-organized brain slices. And the therapeutic effect was evaluated by co-culture of hADSCs and 6-OHDA-constructed brain slice. Within the analysis of hADSCs' exocrine proteins through RNA-seq, Human protein cytokine arrays and label-free quantitative proteomics, key extracellular factors were identified in hADSCs secretion environment.The degeneration of DA neurons and apoptosis were measured in PD samples in vivo and vitro models, and the beneficial effects were evaluated through quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot,Fluoro-Jade C, Tunel assay and immunofluorescence analysis. Results In this study, we discovered that hADSCs protected the dopaminergic (DA) neurons in vivo and vitro models.we identified Pentraxin3 (PTX3) as a key extracellular factor in hADSCs secretion environment. Moreover, we found that human recombinant Pentraxin3 (rhPTX3) treatment could rescue the physiological behaviour of the PD mice in-vivo, as well as prevent DA neurons from death and increase the neuronal terminals in the Ventral tegmental area (VTA) + substantia nigra pars compacta (SNc) and striatum (STR) on the PD brain slices in-vitro. Furthermore, within testing on the pro-apoptotic markers of PD mice brain following the treatment of rhPTX3, we found that rhPTX3 can prevent the apoptosis and the degeneration of DA neurons. Conclusions Overall, the current study investigated that PTX3, a hADSCs secreted protein, played a potential role in protecting the DA neurons from apoptosis and degeneration in PD progression as well as improving the motor performances in PD mice to give a possible mechanism of how hADSCs works in the cell replacement therapy in PD. Importantly, our study also provided potential translational implications for the development of PTX3-based therapeutics in PD.

Inactivation of p38 MAPK Extends Self-Renewal Capacity of Haematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 265-265
Author(s):  
Keisuke Ito ◽  
Atsushi Hirao ◽  
Fumio Arai ◽  
Sahoko Matsuoka ◽  
Keiyo Takubo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
P38 Mapk ◽  
Cell Population ◽  
Critical Role ◽  
Dependent Manner ◽  
Self Renewal ◽  
Haematopoietic Stem Cells ◽  
P38 Inhibitor

Abstract Haematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is critical for maintenance of haematopoietic homeostasis. Here we show that activation of p38 MAPK limits lifespan of HSCs in response to increasing levels of reactive oxygen species (ROS) in vivo. Although normal quiescent HSCs maintain a low level of oxidative stress, an increase in ROS was observed in HSCs after transplantation as well as in aged mice. In vitro treatment with BSO (Buthionine sulfoximine), which depletes intra-cellular glutathion, increased ROS (H2O2) level in immature hematopoietic cell population, c-kit+Sca1+Lin- (KSL) cells, in a dose-dependent manner. Low dose concentration of BSO suppressed reconstitution capacity of HSCs, whereas higher concentration did not affect progenitors. These data indicate that HSCs are much more sensitive to increased ROS than progenitors and are consistent with our previous results from Atm−/− mice in which ROS level is elevated in vivo. Here we focused on MAPKs for the stem cell dysfunction since it has been shown that several MAPKs are activated in response to ROS. We evaluated effects of MAPK inhibitors for p38, JNK or ERK in incubation of KSL cell with BSO. p38 inhibitor (SB203580), neither JNK nor ERK inhibitor, restored reconstitution capacity of HSCs after transplantation, suggesting that activation of p38 may contributes to defect of stem cell function in vivo. To address the question, we evaluated p38 activation in Atm−/− BM cells by immunohistochemistry. Surprisingly, p38 protein was phosphorylated only in KSL cells, but not other more differentiated cell populations, despite that the ROS levels were comparable among the cell population of mice. In response to activation of p38, p16INK4a was up-regulated only in KSL cells. The data indicates a possibility that stem cell-specific p38 activation negatively regulates self-renewal of HSCs. We then investigated a role of p38 activation on self-renewal of HSCs in vivo. When p38 inhibitor was intraperitoneally administered both before and after BMT, the level of repopulation achieved was comparable to that of the wild-type. Furthermore, Atm−/− mice that received long-term p38 inhibitor treatment did not show either anemia, a decrease in progenitor colony-forming capacity, or reduced frequencies of stem cell subsets. These data demonstrate that the activation of p38 present in HSCs promotes the exhaustion of stem cell pool in response to elevation of ROS. It has been proposed that aging is driven in part by a gradual depletion of stem cell functional capacity. There are evidences that inappropriate production of oxidants is connected to aging and life span. We propose a possibility that p38 activation in response to ROS plays a critical role for limit of stem cell capacity.

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