CD133+ Purified Hematopoietic Stem Cells in Co-Culture with Mesenchymal Stromal Cells - The Cell to Cell Contact Matters.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1421-1421
Author(s):  
Nael Alakel ◽  
Douhui Jing ◽  
Martin Bornhaeuser ◽  
Gerhard Ehninger ◽  
Rainer Ordemann
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Cell Fraction ◽  
Adherent Cell ◽  
Cell Contact ◽  
Adherent Cells ◽  
Facs Analysis ◽  
Hematopoietic Stem ◽  
Migration Capacity

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became aware that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD133 purified HSC expansion and differentiation in terms of phenotype, (CXCR4) chemokine receptor expression, migration capacity and clonogenicity. After one week of co-culture with MSC adherent and non-adherent HSC were isolated and analyzed separately. As expected in co-culture experiments with MSC a significant higher expansion of TNC was observed in comparison to the expansion culture without MSC (cytokine driven), 37±6 fold vs. 19±3 fold, p<0.001. However the co-culture expansion was accompanied by a significant increase of differentiation and maturation of the progenitor cells. Therefore we were focusing on the adherent cell fraction of the co-culture system. According to FACS analysis results, adherent HSC in the co-culture display a more immature phenotype than non adherent cells (CD34+CD38−). Next colony assays and LTC-IC were performed showing that the adherent cell fraction contained a significant higher proportion of primary and secondary CFU-C, indicating that this cell fraction contains the majority of clonogenic HSC. The CXCR4 - SDF-1 axis plays an important rule in homing, mobilization and engraftment of the hematopoietic stem cells. Therefore we looked for CXCR4 expression in the two cell fractions using FACS analysis. The adherent cells expressed consistently and significantly more CXCR4 than the non adherent cells; 43±4% vs 15±2%, p<0.001. Data could be confirmed by performing western blot. Due to this data we evaluated the capacity of HSC migration toward SDF-1 gradient using transwell migration assay. The migration capacity of the adherent cells was significantly higher than that of the non adherent cell fraction correlating with the CXCR4 expression; 13±1% vs. 6±1%, p<0.001. We checked the expression of different cell adhesion molecules by FACS analysis. CD44 and CD49e (integrin α5) were significantly higher expressed in adherent cells comparing with non-adherent cells. Our data indicate that the cell-to-cell interaction mediated impact on CD133+ selected HSC leads to an enhanced clonogenic capacity and migratory potential. Further experiments are needed to investigate engraftment and NOD/SCID repopulation potential of the adherent and non-adherent cells after ex-vivo expansion.

Localization of Hematopoietic Stem Cells in Coculture with Mesenchymal Stromal Cells Impacts on Phenotype and Cell Cycle Status

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4774-4774
Author(s):  
Duohui Jing ◽  
Nael Alakel ◽  
Fernando Fierro ◽  
Katrin Mueller ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Stromal Cells ◽  
The Other ◽  
Cell Contact ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Cell Fractions

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p&lt;0.001. The data indicate the facilitating impact of MSC on HSC in performing mitosis which is however depending on the location of interaction. When HSC are released into supernatant (non adherent cells) or covered by MSC, G2 phase was significantly down-regulated. Next we studied the proliferation capacity of the separate cell fractions. Consistent with the data of cell cycle, cell number of phase-bright faction increased much faster than the other two fractions during the first 4 days suggesting that the MSC surface in vitro is the predominant location of HSC proliferation. Next we investigated the phenotype of HSC. According to FACS analysis results (CD34+CD38-) phase-dim cells revealed a more immature phenotype in comparison to the non adherent cells and the phase-bright cells. During the first four days 80% of phase-dim cells remained CD34+CD38-, while cells of the phase-bright- and the non adherent fraction exhibited a significant more mature phenotype. Performing cell division tracking using CFSE we were able to show that over time number of divisions of phase-dim cells were significantly diminished in comparison to the other two cell fractions in co-cultures. In addition, phase-dim cells started to lose CD34 at the 7th generation, while non-adherent and phase-bright cells already lost CD34 at the 4th generation. These data suggest that “stemness” of HSC was rather preserved in the cell fraction which was covered by MSC monolayer than in the cell fraction on the surface of MSC. In conclusion we demonstrate HSC in distinct locations in vitro showing different behaviors in terms of phenotype and proliferation. It becomes evident that not only the cell to cell contact matters but also the localization of contact. Further experiments are needed to investigate NOD/SCID repopulation potential of the different cell fractions.

scholarly journals Proliferation and differentiation of hematopoietic stem cells in long-term cultures of adult hamster spleen.

1982 ◽  
Vol 155 (5) ◽  
pp. 1370-1384 ◽  
Author(s):  
E A Arnold ◽  
I Katsnelson ◽  
G J Hoffman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Horse Serum ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Basic Medium ◽  
Adherent Cell ◽  
Hematopoietic Stem ◽  
Adult Hamster

We have found that in liquid cultures of spleen cells of adult Syrian hamsters of the F1D strain, the hematopoietic microenvironment is adequate to sustain proliferation of splenic stem cells for periods of greater than 4 mo, and permits granulocytic, monocytic, and megakaryocytic differentiation without secondary repopulation or addition of exogenous growth factors to the basic medium of RPMI 1640 plus 20% horse serum. Intimate topographical relations are established between spleen stromal cells and hematopoietic cell components of the culture is adherent "cell-producing" islets. Some of these islets are associated with multiple hematopoietic cell types such as myeloid, monocytic, and megakaryocytic cells. Other islets are associated with a single cell type such as megakaryocytes, which suggests a limited potential of some adherent stromal cells to direct the differentiation of precursor cells. Cultures of this type provide a simple and convenient model for investigation of the mechanisms controlling differentiation of hematopoietic stem cells, not only for granulocytic and monocytic cells, but for megakaryocytic cells as well.

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

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.

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

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

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

2015 ◽  
Vol 39 (10) ◽  
pp. 1099-1110 ◽  
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

scholarly journals Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vivo

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Futoshi Hashimoto ◽  
Kikuya Sugiura ◽  
Kyoichi Inoue ◽  
Susumu Ikehara
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Major Histocompatibility Complex ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Graft Failure ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Histocompatibility Complex ◽  
Colony Forming Units

Graft failure is a mortal complication in allogeneic bone marrow transplantation (BMT); T cells and natural killer cells are responsible for graft rejection. However, we have recently demonstrated that the recruitment of donor-derived stromal cells prevents graft failure in allogeneic BMT. This finding prompted us to examine whether a major histocompatibility complex (MHC) restriction exists between hematopoietic stem cells (HSCs) and stromal cells. We transplanted bone marrow cells (BMCs) and bones obtained from various mouse strains and analyzed the cells that accumulated in the engrafted bones. Statistically significant cell accumulation was found in the engrafted bone, which had the same H-2 phenotype as that of the BMCs, whereas only few cells were detected in the engrafted bones of the third-party H-2 phenotypes during the 4 to 6 weeks after BMT. Moreover, the BMCs obtained from the MHC-compatible bone showed significant numbers of both colony-forming units in culture (CFU-C) and spleen colony-forming units (CFU-S). These findings strongly suggest that an MHC restriction exists between HSCs and stromal cells.

scholarly journals CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages

Blood ◽  
2001 ◽  
Vol 97 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Sahoko Matsuoka ◽  
Yasuhiro Ebihara ◽  
Ming-jiang Xu ◽  
Takefumi Ishii ◽  
Daisuke Sugiyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Developmental Stages ◽  
Marrow Cell ◽  
Cell Activity ◽  
Cell Fraction ◽  
Hematopoietic Stem ◽  
Cd34 Antigen ◽  
Cd34 Expression

Abstract The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin−c-Kit+ long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin−c-Kit+CD34− marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin−c-Kit+ CD34high HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin−c-Kit+CD34− cells after 16 weeks, indicating that adult Lin−c-Kit+CD34− HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.

Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vitro

Stem Cells ◽  
2001 ◽  
Vol 19 (1) ◽  
pp. 46-58 ◽  
Author(s):  
Kikuya Sugiura ◽  
Hiroko Hisha ◽  
Junji Ishikawa ◽  
Yasushi Adachi ◽  
Shigeru Taketani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Major Histocompatibility Complex ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Histocompatibility Complex

scholarly journals Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vivo

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Futoshi Hashimoto ◽  
Kikuya Sugiura ◽  
Kyoichi Inoue ◽  
Susumu Ikehara
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Major Histocompatibility Complex ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Graft Failure ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Histocompatibility Complex ◽  
Colony Forming Units

Abstract Graft failure is a mortal complication in allogeneic bone marrow transplantation (BMT); T cells and natural killer cells are responsible for graft rejection. However, we have recently demonstrated that the recruitment of donor-derived stromal cells prevents graft failure in allogeneic BMT. This finding prompted us to examine whether a major histocompatibility complex (MHC) restriction exists between hematopoietic stem cells (HSCs) and stromal cells. We transplanted bone marrow cells (BMCs) and bones obtained from various mouse strains and analyzed the cells that accumulated in the engrafted bones. Statistically significant cell accumulation was found in the engrafted bone, which had the same H-2 phenotype as that of the BMCs, whereas only few cells were detected in the engrafted bones of the third-party H-2 phenotypes during the 4 to 6 weeks after BMT. Moreover, the BMCs obtained from the MHC-compatible bone showed significant numbers of both colony-forming units in culture (CFU-C) and spleen colony-forming units (CFU-S). These findings strongly suggest that an MHC restriction exists between HSCs and stromal cells.

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