scholarly journals 167 ISOLATION AND COMPARATIVE PROFILING OF HUMAN ADIPOSE-DERIVED ADULT STEM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 234
Author(s):  
A. Boquest ◽  
A. Shahdadfar ◽  
K. Fronsdal ◽  
J. Brinchmann ◽  
P. Collas
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Cell Signaling ◽  
Proliferative Capacity ◽  
Stromal Stem Cells

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. However, despite their potential impact in the field of regenerative medicine, little is known about the biology of stromal stem cells prior to culture. After removing adipocytes and erythrocytes from collagenase digested human adipose tissue, we identified two cell populations using flow cytometry which shared expression of stem cell markers SH2 and CD34, but lacked the phenotypic characteristics of leukocytes (CD45−). However, they were found to be discernible based on CD31 expression, a marker for endothelial cells. Using CD31 conjugated magnetic beads, we separated these cells (CD45-CD31− and CD45-CD31+) from three patients and compared global gene expression profiles using an Affymetrix platform. The prominant feature of CD45-CD31+ cells was the up-regulation of genes associated with endothelial cells. By contrast, CD45-CD31− cells were found to overexpress transcripts involved in cell cycle quiescence and cell signaling elements including those of the WNT pathway thought to be important for maintaining the stem cell state. Upon culture in DMEM/F12 with 20% FCS, only CD45-CD31− cells were capable of adhering to plastic and forming colonies. These cells with fibroblastic morphology met the key criterion of stem cells, the ability to proliferate while retaining the capacity to differentiate into mature tissues. Under appropriate inductive conditions, they were found to exclusively form bone, cartilage, adipose and neuronal-like tissues in vitro. Clonal cell lines generated from individually cultured CD45-CD31− cells displayed multilineage and proliferative capacity, validating our conclusion that they are true stem cells and not simply committed progenitors. We then undertook extensive comparative profiling of CD45-CD31− cells with their cultured counterparts to examine changes that stromal stem cells undergo during culture. Except for the disappearance of CD34, flow cytometry analysis using 52 antibodies revealed little change in cell surface phenotype as a result of culture. However, comparative global gene profiling revealed extensive down-regulation of many genes during culture. These included cell cycle arresting genes, as expected, and genes encoding elements involved in cell signaling including those belonging to the tumor necrosis factor, interleukin, transforming growth factor and chemokine families. The consequences of these changes remain unknown, but ultimately may affect the potential use of adipose tissue stem cells in regenerative medicine.

scholarly journals Effect of Placental Extract on Omentum Mesenchymal Stem Cells Differentiation in NMRI Mice

2017 ◽  
Vol 7 (1) ◽  
pp. 176
Author(s):  
Maryam Sadat Nezhadfazel ◽  
Kazem Parivar ◽  
Nasim Hayati Roodbari ◽  
Mitra Heydari Nasrabadi
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Control Group ◽  
Fat Cell ◽  
Nmri Mice ◽  
Differentiated Cells ◽  
Placental Extract

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

scholarly journals Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Patrick C. Baer ◽  
Helmut Geiger
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Cultured Cells ◽  
Current Knowledge ◽  
High Plasticity ◽  
Culture Methods ◽  
Tissue Localization ◽  
Stromal Stem Cells

Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs) yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneityin vitro, and the lack of standardization in isolation and culture methods.

scholarly journals Changes in ionizing radiation dose rate affect cell cycle progression in adipose derived stem cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250160
Author(s):  
Matthew Rusin ◽  
Nardine Ghobrial ◽  
Endre Takacs ◽  
Jeffrey S. Willey ◽  
Delphine Dean
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Radiation Therapy ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Ionizing Radiation ◽  
Dose Rate ◽  
Cell Types ◽  
High Energy ◽  
Adipose Derived Stem Cells

Biomedical use of radiation is utilized in effective diagnostic and treatment tools, yet can introduce risks to healthy tissues. High energy photons used for diagnostic purposes have high penetration depth and can discriminate multiple tissues based on attenuation properties of different materials. Likewise, the ability to deposit energy at various targets within tumors make the use of photons effective treatment for cancer. Radiation focused on a tumor will deposit energy when it interacts with a biological structure (e.g. DNA), which will result in cell kill should repair capacity of the tissue be overwhelmed. Likewise, damage to normal, non-cancerous tissues is a consequence of radiation that can lead to acute or late, chronic toxicity profiles. Adipose derived stem cells (ADSCs) are mesenchymal stem cells that have been proven to have similar characteristics to bone marrow derived stem cells, except that they are much easier to obtain. Within the body, ADSCs act as immunomodulators and assist with the maintenance and repair of tissues. They have been shown to have excellent differentiation capability, making them an extremely viable option for stem cell therapies and regenerative medicine applications. Due to the tissue ADSCs are derived from, they are highly likely to be affected by radiation therapy, especially when treating tumors localized to structures with relatively high ADSC content (eg., breast cancer). For this reason, the purpose behind this research is to better understand how ADSCs are affected by doses of radiation comparable to a single fraction of radiation therapy. We also measured the response of ADSCs to exposure at different dose rates to determine if there is a significant difference in the response of ADSCs to radiation therapy relevant doses of ionizing radiation. Our findings indicate that ADSCs exposed to Cesium (Cs 137)-gamma rays at a moderate dose of 2Gy and either a low dose rate (1.40Gy/min) or a high dose rate (7.31Gy/min) slow proliferation rate, and with cell cycle arrest in some populations. These responses ADSCs were not as marked as previously measured in other stem cell types. In addition, our results indicate that differences in dose rate in the Gy/min range typically utilized in small animal or cell irradiation platforms have a minimal effect on the function of ADSCs. The potential ADSCs have in the space of regenerative medicine makes them an ideal candidate for study with ionizing radiation, as they are one of the main cell types to promote tissue healing.

scholarly journals Isolation and Transcription Profiling of Purified Uncultured Human Stromal Stem Cells: Alteration of Gene Expression after In Vitro Cell Culture

2005 ◽  
Vol 16 (3) ◽  
pp. 1131-1141 ◽  
Author(s):  
Andrew C. Boquest ◽  
Aboulghassem Shahdadfar ◽  
Katrine Frønsdal ◽  
Olafur Sigurjonsson ◽  
Siv H. Tunheim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Adipose Tissue ◽  
Human Adipose Tissue ◽  
Histocompatibility Complex ◽  
Neural Tissues ◽  
Organ Specific ◽  
Stromal Stem Cells ◽  
Two Populations

Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal components were up-regulated. CD31+ cells did not proliferate in vitro. CD45-CD34+CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.

scholarly journals METTL3-mediated m6A modification regulates cell cycle progression of dental pulp stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Haiyun Luo ◽  
Wenjing Liu ◽  
Yanli Zhang ◽  
Yeqing Yang ◽  
Xiao Jiang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Fate ◽  
Dental Pulp ◽  
Interaction Network ◽  
Dental Pulp Stem Cells ◽  
Tunel Staining

Abstract Background Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of m6A methylation in DPSCs. Methods m6A immunoprecipitation with deep sequencing (m6A RIP-seq) demonstrated the features of m6A modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by β-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and m6A RIP-qPCR was used to confirm METTL3-mediated m6A methylation. Results Here, m6A peak distribution, binding area, and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of m6A RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated m6A methylation in DPSCs. Conclusions These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and served new insight into stem cell-based tissue engineering.

scholarly journals Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1481-1481
Author(s):  
Yueksel Filik ◽  
Karin Bauer ◽  
Barbara Peter ◽  
Emir Hadzijusufovic ◽  
Georg Greiner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Induced Resistance ◽  
Research Funding ◽  
Mtor Inhibitor ◽  
Pi3 Kinase

Abstract Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. Disclosures Hoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

scholarly journals Fundamentals of Stem Cells: Why and How Patients' Own Adult Stem Cells Are the Next Generation of Medicine

2019 ◽  
Author(s):  
Eckhard U. Alt ◽  
Christoph Schmitz ◽  
Xiaowen Bai
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Adult Stem Cells ◽  
Point Of Care ◽  
Appropriate Technology ◽  
Differentiation Potential ◽  
Resident Stem Cells ◽  
New Generation

Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold certain promise for regenerative medicine. This paper is intended to clarify and facilitate the understanding, development and adoption of regenerative medicine in general and specifically of therapies based on unmodified, autologous adipose-derived regenerative cells (UA-ADRCs). To this end, results of landmark experiments on stem cells and stem cell therapy performed in the labs of the authors are summarized, the most intriguing of which are the following: (i) vascular associated mesenchymal stem cells (MSCs) can be isolated from different organs (adipose tissue, heart, skin, bone marrow and skeletal muscle) and differentiated into ectoderm, mesoderm and endoderm, providing significant support for the hypothesis of the existence of a small, ubiquitously distributed, universal vascular associated stem cell with full pluripotency; (ii) the orientation and differentiation of MSCs are driven by signals of the respective microenvironment; and (iii) these stem cells irrespective of the tissue origin exhibit full pluripotent differentiation potential without any prior genetic modification or the need for culturing. They can be obtained from a small amount of adipose tissue when using the appropriate technology for isolating the cells, and can be harvested from and re-applied to the same patient at the point of care without the need for complicated processing, manipulation, culturing, expensive equipment, or repeat interventions. These findings demonstrate the potential of UA-ADRCs for triggering the development of an entire new generation of medicine for the benefit of patients and of healthcare systems.

scholarly journals Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2824 ◽  
Author(s):  
Bong-Sung Kim ◽  
Pathricia V. Tilstam ◽  
Katrin Springenberg-Jung ◽  
Arne Hendrick Boecker ◽  
Corinna Schmitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Subcutaneous Adipose Tissue ◽  
Stem Cell Markers ◽  
Adipose Derived Stem Cells ◽  
Cell Markers ◽  
Adipose Tissue Macrophages ◽  
Subcutaneous Adipose

Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds.

scholarly journals Adipose-Derived Stem Cells for Future Regenerative System Medicine

2012 ◽  
Vol 4 (2) ◽  
pp. 59
Author(s):  
Yani Lina ◽  
Andi Wijaya
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Adipose Tissue ◽  
Regenerative Medicine ◽  
Adult Stem Cells ◽  
Stromal Vascular Fraction ◽  
Human Adipose Tissue ◽  
Lytic Enzymes ◽  
Stromal Stem Cells

BACKGROUND: The potential use of stem cell-based therapies for repair and regeneration of various tissues and organs offers a paradigm shift that may provide alternative therapeutic solutions for a number of disease. Despite the advances, the availability of stem cells remaining a challenge for both scientist and clinicians in pursuing regenerative medicine. CONTENT: Subcutaneous human adipose tissue is an abundant and accessible cell source for applications in tissue engineering and regenerative medicine. Routinely, the adipose issue is digested with collagenase or related lytic enzymes to release a heterogeneous population for stromal vascular fraction (SVF) cells. The SVF cells can be used directly or can be cultured in plastic ware for selection and expansion of an adherent population known as adipose-derived stromal/stem cells (ASCs). Their potential in the ability to differentiate into adipogenic, osteogenic, chondrogenic and other mesenchymal lineages, as well in their other clinically useful properties, includes stimulation of angiogenesis and suppression of inflammation.SUMMARY: Adipose tissue is now recognized as an accessible, abundant and reliable site for the isolation of adult stem cels suitable for the application of tissue engineering and regenerative medicine applications. The past decade has witnessed an explosion of preclinical data relating to the isolation, characterization, cryopreservation, differentiation, and transplantation of freshly isolated stromal vascular fraction cells and adherent, culture-expanded, adipose-derived stromal/stem cells in vitro and in animal models.KEYWORDS: adipose tissue, adult stem cells, regenerative medicine, mesenchymal stem cells

Comparison Of Autologous Microparticles and Fucosyltransferase-7 For Adhesion Improvement Of Hematopoietic Stem Cells To Bone Marrow Endothelial Cells In a Microfluidic Flow Chamber

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2000-2000
Author(s):  
Arne Trummer ◽  
Dennis Rataj ◽  
Sonja Werwitzke ◽  
Andreas Tiede
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Flow Chamber ◽  
Sialyl Lewis X ◽  
Study Phase ◽  
Hematopoietic Stem ◽  
Microfluidic Flow

Abstract The improvement of graft function and time to engraftment might help to reduce infection-related mortality in stem cell transplantation (SCT). While the concept of stem cells fucosylation for accelerated engraftment has already reached clinical study phase (for cord blood transplantation; NCT01471067), own previous work has shown an association between engraftment time and circulating microparticles bearing P-Selectin and P-Selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 contains the sialyl Lewis x (CD15s) antigen that requires fucosylation for optimal binding of P- and E-Selectin on endothelial cells. We therefore hypothesized that addition of microparticles (MP) might enhance adhesion of human stem cells (HSC) to bone marrow endothelial cells and that MP might have synergistic effects in combination with stem cell fucosylation. HSC were obtained from apheresis products of allogeneic donors, purified by Ficoll and magnetic bead separation for CD34, stained with calcein AM and perfused through an automated microfluidic flow chamber (Bioflux 200, Fluxionbio, USA) covered with a confluent layer of an immortalized human bone marrow endothelial cell line (HBMEC). Photos (and videos) were taken using a fluorescence microscope at start, 5 min and 10 min and analyzed for adherent HSC across the whole chamber (about 1.5 sqmm) using ImageJ software. Autologous MP were generated by addition of calcimycin to apheresis and isolation of MP by centrifugation. For control experiments, one part of the MP solution was passed through a 0,2µm-filter to remove MP. MP concentration (mean: 1362/µl) was assessed by detection of Annexin V binding in flow cytometry, using TrucountBeads® for quantification. Fucosylation was performed by 1h incubation of isolated CD34+ stem cells with GDP-fucose and fucosyltransferase 7 (FUT7). Successful fucosylation was controlled by CD15s staining of HSC in flow cytometry. Results of seven experiments (in duplicate) demonstrated the highest number of adherent HSC in the MPpositiv/FUT7negativ preparation (median: 32 HSC/sqmm; range: 15-78), followed by MPpositiv/FUT7positiv (30 HSC/sqmm; range: 16-38), MPnegativ/FUT7positiv (median: 25/sqmm; range: 11-27) and MPnegativ/FUT7negativ (20 HSC/sqmm; range: 0-22). Comparison of the MPpositiv/FUT7negativ and MPnegativ/FUT7negativ as well as the MPpositiv/FUT7positiv and MPpositiv/FUT7negativ preparations showed statistically significant differences in Wilcoxon rank test (p<.05) while comparison of MPpositiv/FUT7positiv vs. MPnegativ/FUT7positiv and MPnegativ/FUT7positiv vs. MPnegativ/FUT7negativ preparations did not. In summary, these results demonstrate that MP can improve HSC adhesion to bone marrow endothelial cells similar to fucosylation. The effect of fucosylation on HSC adhesion appears to be mediated by MP. However, there is not a synergistic effect between MP and fucosylation. Disclosures: No relevant conflicts of interest to declare.

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