Direct contact with mesenchymal stromal cells affects migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex vivo expansion

Abstract Abstract 4694 Background: Quiescent hematopoietic stem cells (HSCs) located in stem cell niches are characterized by a relative resistance to hypoxia. This study is focused primarily on maintainance of the repopulating ability of HSCs in structurally intact BM exposed to anoxia, lack of metabolic substrates and accumulation of metabolic waste products during a period of ischemia at three different temperatures. In the case of a warm ischemia at 37°C, changes in gene expression profile in the whole bone marrow has been also examined. Methods: Murine congenic model C57Bl/6 Ly5.1/Ly5.2 was used in the experiments. Normal mice or mice recovering from a bone marrow damage induced either by cyclophosphamide or a sublethal irradiation were sacrified. Their BM was maintained in intact femurs at 37°C for different time periods up to 6 hours. For normal bone marrow, exposure to ischemia at 20°C and 4°C was also used for up to 20 and 48 hours, respectively. Afterwards, bone marrow cells were harvested and cells corresponding to a half of the femur were transplanted to sublethally (6 Gy) irradiated recipients in a competitive repopulation assay. Resulting chimerism was examined up to 6 months after transplantation to test for STRCs and LTRCs (Short and Long Term Repopulating Cells). Subpopulations of erythropoietic (Ter119+), B-lymphopoietic (B220+), granulo- and monocytopoietic (Gr-1/Mac+), and LSK (Lin-Sca-1+c-Kit+) bone marrow cells were analyzed for dead cells and apoptosis. Total RNA was isolated from bone marrow exposed to warm ischemia ranging 0 to 4 hours and dynamics of changes in its gene expression profile was determined by Illumina MouseRef8 BeadChip. Results: Repopulating ability of ischemic BM was fully preserved for 2 hour of the warm (37°C) ischemia and for 6 hours and 8 hours of 20°C and 4°C ischemia, respectively. There was no difference between STRCs and LTRCs in survival. STRCs and LTRCs from the bone marrow collected 2 days or 5 days after a single dose of cyclophosphamide exposed to warm ischemia showed decreased repopulating ability in comparison with those of normal mice. STRCs significantly prevailed over LTRCs in bone marrow collected 20 days after a sublethal irradiation and showed increased sensitivity to warm ischemia. B220+ cells were the most sensitive cells of the bone marrow to warm ischemia, LSK and Ter119 cells being the most resistant ones. Gene expression profile in bone marrow exposed to warm ischemia changed progressively over time. Despite the highly unfavorable metabolic conditions, hypoxia and lack of energy, a set of overexpressed genes equaled in number the one inhibited. Conclusions: HSCs exposed to warm or cold ischemia maintain their repopulating ability for a considerable time. Bone marrow ischemia activates specific gene expression in paralel with supression of others. Supported by projects LC06044, MSM 0021620806 and the grant SVV-2010-254260507. Disclosures: No relevant conflicts of interest to declare.

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A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200124115444 ◽

2020 ◽

Vol 15 (3) ◽

pp. 250-262

Author(s):

Maryam Islami ◽

Fatemeh Soleimanifar

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Umbilical Cord ◽

Stromal Cells ◽

Ex Vivo ◽

Hematologic Malignancies ◽

Future Directions ◽

Hematopoietic Stem ◽

Efficient Procedure ◽

Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

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Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

Stem Cells Translational Medicine ◽

10.1002/sctm.21-0130 ◽

2021 ◽

Author(s):

Valentina Orticelli ◽

Andrea Papait ◽

Elsa Vertua ◽

Patrizia Bonassi Signoroni ◽

Pietro Romele ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem

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The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

Cell Biology International ◽

10.1002/cbin.10483 ◽

2015 ◽

Vol 39 (10) ◽

pp. 1099-1110 ◽

Cited By ~ 4

Author(s):

Iordanis Pelagiadis ◽

Eftichia Stiakaki ◽

Christianna Choulaki ◽

Maria Kalmanti ◽

Helen Dimitriou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem

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Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells

Indian Journal of Hematology and Blood Transfusion ◽

10.1007/s12288-015-0545-y ◽

2015 ◽

Vol 32 (1) ◽

pp. 67-77 ◽

Cited By ~ 2

Author(s):

Naser Amirizadeh ◽

Arezoo Oodi ◽

Roya Mehrasa ◽

Mahin Nikougoftar

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Umbilical Cord Blood ◽

Stromal Cells ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Cytokine Milieu

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A secreted and LIF-mediated stromal cell–derived activity that promotes ex vivo expansion of human hematopoietic stem cells

Blood ◽

10.1182/blood.v95.6.1957 ◽

2000 ◽

Vol 95 (6) ◽

pp. 1957-1966 ◽

Cited By ~ 40

Author(s):

Chu-Chih Shih ◽

Mickey C.-T. Hu ◽

Jun Hu ◽

Yehua Weng ◽

Paul J. Yazaki ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Cell Expansion ◽

Culture System ◽

Stromal Cell ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Stem Cell Expansion

Abstract The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion.

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Gene-Chip Analysis of Cord Blood Derived CD34+ Cells Expanded on Bioartificial Materials.

Blood ◽

10.1182/blood.v104.11.4131.4131 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4131-4131

Author(s):

Joachim Oswald ◽

Christine Steudel ◽

Katrin Salchert ◽

Christian Thiede ◽

Gerhard Ehninger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Fibrillar Collagen ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Colony Forming Units

Abstract Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen. The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology. Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.

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