Your browser is out-of-date!

Update your browser to view this website correctly. Update my browser now

×

Discipline
Biological
Keywords
Bone Marrow-Mimetic Extracellular Matrix Scaffold
Erythroid Commitment
Integrin Beta3
Observation Type
Standalone
Nature
Standard Data
Submitted
Aug 26th, 2017
Published
Oct 12th, 2017
  • Abstract

    Within the bone marrow (BM) microenvironment erythroid development of hematopoietic stem and progenitor cells (HSPCs) is regulated by extracellular matrix (ECM) anchored factors like fibronectin (FN), derived from mesenchymal stromal cells (MSCs). Thus, effective ex-vivo erythroid cell development in translational medicine could benefit from the addition of naïve ECM preparations. Native BM-mimetic ECMs derived from MSCs were obtained by a secretome anchoring technique based on reactive copolymer thin film chemistry. The stabilization process is initiated by covalent binding of FN to glass surfaces, coated with 3-Aminopropyl-dimethyl-silyl and poly-(octadecene-alt-maleic anhydride). After MSC decellularization ECMs were used as HSPC culture substrates. Within a 5 day culture period adherent and non-adherent HSPCs upregulate integrin β3 (ITGβ3) surface expression. In colony formation assays erythroid potential of clonogenic ECM cultured cells was increased, which was even more pronounced when plating CD34+ITGβ3+ sorted HSPCs. Thus, ECM supported ex-vivo culture seems to promote erythroid differentiation.

  • Figure
  • Introduction

    Within the bone marrow (BM), hematopoietic stem and progenitor cells (HSPCs) reside in close proximity to mesenchymal stromal cells (MSCs) tightly anchored to an extracellular matrix (ECM) scaffold; a highly crosslinked, insoluble fibrillary structure assembled of multiple proteins including fibronectin, collagen, proteoglycans, and laminin. Besides structural integrity, ECM can store and release growth factors and cytokines. Moreover, the ECM composition provides topographical and biomechanical features, which can regulate HSPC fate and determine tissue specificity. Cell-ECM communication is a highly dynamic process; cells modulate the ECM structure constantly, which thereby allows for cell migration and growth factor release.

    A cell-secretome anchoring technique can be used to generate naïvely constituted, BM-mimicking ECM scaffolds derived from MSCs. It was reported that HSPCs cultured on those ECM scaffolds derived from a MSC line (SCP-1) can sense these ECM features via integrin β3 (ITGβ3) adhesion receptor, which is associated with erythroid lineage commitment.

    Following this line, ECM proteins exhibit multiple binding sites for colony-stimulating factors (CSFs), which can trigger myeloid lineage commitment. Additionally, ECM proteins like fibronectin (FN) were found to be essential for erythropoiesis and pro-erythroid growth factors like IL-3 and BMP4 are incorporated in ECM scaffolds. Therefore, we asked whether BM-mimetic ECM scaffolds might trigger erythropoiesis when culturing HSPC.

  • Objective

    As ITGβ3 expression/activation on HSPCs is known to drive erythroid commitment. We aimed at identifying a potent erythroid lineage bias when using SCP-1 cell-derived ECM scaffolds to culture and expand primary human CD34+ HSPCs.

  • Results & Discussion

    Within the BM, HSPCs reside anchored to stromal cell-derived ECM proteins. In order to mimic this interaction ex-vivo, we produced ECM scaffolds derived from SCP-1 cells. These cells were shown to have a naïve, human MSC phenotype. SCP-1 cells were cultured for up to 10 days on functionalized glass slides, which immobilize secreted proteins by a covalent linkage to glass surfaces. For ECM protein binding bioactive fibronectin (FN) served as a substrate, which was covalently linked to the glass surface by previous 3-Aminopropyl-dimethyl-silyl and poly-(octadecene-alt-maleic anhydride) coating. To decellularize the secreted scaffold SCP-1 cells were treated with warm double distilled water supplemented with 20 mM ammonium hydroxide under constant agitation. Cell debris was removed by multiple washing steps (Fig. 1A). This bio-reactive thin film chemistry allowed for stabilization of secreted ECM and kept protein constitution in a naïve state. It was reported recently that 20% of HSPCs isolated from G-CSF mobilized PB adhere to SCP-1 derived ECM scaffolds. However, 80% of HSPCs remained non-adherent in the culture. Accordingly, the cells were termed attached (AT) or supernatant (SN) cells. ECM scaffolds were identified as highly supportive for HSPC proliferation with a 2-fold expansion after 5 days in culture. Adhesion was found to initiate ITGb3 surface expression on up to 60% of ECM cultured HSPCs. However, ITGb3 expression could also represent a marker for lineage commitment, since it has been shown to be predominantly expressed in megakaryopoiesis and erythropoiesis.

    To identify HSPC myeloid commitment in standard plastic culture dish (PCD) suspension culture as a control or ECM culture we designed the experimental set-up as outlined in figure 1A to C. CD34+ HSPCs were isolated from G-CSF mobilized PB, cultured for 5 days on PCD or ECM and re-isolated for CD34 expression. We obtained 3 CD34+ cell fractions, PCD-cells, AT-cells and SN-cells and compared their clonogenicity and differentiation potential using myeloid colony forming unit assay (CFU-GEMM) to freshly isolated HSPCs as a second control (Fig. 1B). In a subsequent set of experiments, FACS was used to separate freshly isolated HSPCs, AT-cells and SN-cells into CD34+ITGb3+ and CD34+ITGb3- fraction (Fig. 1C). We compared colony forming unit-erythrocyte (CFU-E) to other myeloid colonies, including CFU-granulocyte (CFU-G), CFU-granulocyte/macrophage (CFU-GM) and CFU-macrophage (CFU-M). Interestingly, the cumulative number of CFUs was not altered when analyzing freshly isolated CD34+ cells compared to CD34+ cells cultured either on PCD or ECM (Fig. 1D). This shows the maintenance of myeloid progenitors over 5 days of culture independent of culture conditions. However, regarding the distribution of erythroid colonies, ECM cultured HSPCs, either AT- or SN-cells, exhibited a significant increase in CFU-Es, as compared to freshly isolated- and PCD cultured cells (Fig. 1E). Vice versa, ECM cultured AT- and SN-cells showed reduced myeloid colony formation regarding CFU-G, CFU-M and CFU-GM (Fig. 1F).

    It has been demonstrated that erythropoiesis requires ITGb1 mediated FN adhesion and ITGb3 expression is a marker for erythroid commitment. To test whether erythroid lineage commitment is associated with ITGb3 expression in our ex-vivo ECM culture, we separated CD34+ITGb3+ and CD34+ITGb3- cells. CD34+ITGb3+ cells exhibit significantly elevated colony formation with approximately 455 ± 33 colonies compared to 214 ± 20 colonies in CD34+ITGb3- cells isolated freshly from PB (Fig. 1G). This finding could partly be explained by a previously described enrichment of long-term repopulating HSPCs in an ITGb3-dependent fashion. However, the amount of CD34+ITGb3+ cells in PB is approximately 2% only. Increased colony formation was also visible for CD34+ITGb3+ SN-cells compared to AT-cells. Here, ITGb3 expression was not associated with increased total colony formation (Fig. 1G) suggesting that adhesion to ECM scaffolds induces rapid cell commitment which might lead to cell detachment. This could also explain the increased amount of CFU-Es in the SN-cell fraction. The number of total CFUs was approximately the same in CD34+ITGb3- cells isolated from PB-, AT- and SN-cells compared to the amount of CFUs of CD34+ isolated cells described above. This suggests that ITGb3 surface expression is negligible in the context of hematopoietic stem cell expansion. Though, when HSPCs were cultured on ECM, CD34+ITGb3+ cells generate mostly CFU-Es, only a few CFU-Gs and neither CFU-Ms nor CFU-GMs, as compared to CD34+ITGb3- cells (Fig. 1H and I). PB-derived, freshly isolated CD34+ITGb3+ cells were found to differentiate into all myeloid lineages but also exhibited a significant enrichment in CFU-Es compared to their CD34+ITGb3- counterpart (Fig. 1H and I). This identifies the rapid induction of ITGb3 expression on HSPCs when cultured on SCP-1 cell-derived ECMs as marker or trigger to erythroid differentiation.

  • Conclusions

    Our findings suggest an association between the induction of ITGb3 surface expression on CD34+ cells in contact cultures on ECM preparations and erythroid lineage commitment. However, the underlying mechanisms and signaling pathways involved need to be carefully elucidated.

    These findings could be of high interest regarding the ex-vivo generation of red blood cell precursors, e.g. when in vitro erythrocyte differentiation from induced pluripotent stem cells is attempted. Our data suggest that differentiation protocols could benefit from the addition of defined ECM preparations in order to increase the efficiency of red blood cell generation.

  • Limitations

    HSPC expansion is rather low. It was shown in the past that HSPC expansion is much higher on decellularized scaffolds compared to our study. However, we used a lower cytokine concentration (2.5 ng/ml SCF, IL-3 and Flt-3 each) compared to other studies. We attempt to prevent HSPC differentiation due to high proliferation to emphasize the ECM-derived lineage commitment effect.

    Erythropoiesis markers like CD235a or CD71 were not analyzed using flow cytometry. Quantifying these markers could be used to assay erythroid progenitors and erythropoiesis in detail.

    CFU-GEMM assay was used to measure progenitor cell function but different colony types (CFU-G, CFU-M, CFU-GM or CFU-GEMM) were not observed individually. Additionally, erythroid colonies were not separated in CFU-E and BFU-E, which would allow for a more detailed examination of differentiation status.

  • Alt. Explanations

    As we found an increase in CD34+ITGb3+ HSPCs on ECM cultures and increased total colony formation of these cells in CFU-GEMM assay, one could interpret these findings as increased stem cell activity; however, CFU-GEMM assay identifies only hematopoietic progenitor activity.

  • Conjectures

    Progenitor commitment with respect to erythropoiesis needs to be clarified by flow cytometry and detailed analyses of CFU-GEMM assays as mentioned above.

    Within the next steps, we plan to identify the connection between ITGb3 expression, ECM adhesion and cell commitment. Using mass spectrometry, we aim at identifying pro-erythropoiesis factors within the SCP-1 cell-derived ECM to elucidate the underlying mechanism of erythroid commitment. Finally, in-vivo experiments could show whether erythroid outgrowth is enhanced in transplant models.

  • Methods

    SCP-1 cell culture

    SCP-1 cell line was generously provided by Matthias Schieker. Cells were cultured in low-glucose DMEM (Life technology, USA) with 10% FCS (Biochrom, USA) at 37°C and 5% CO2 in a humidified incubator. Cells were passaged once a week and the medium was changed twice a week.

    Generation of surface immobilized SCP-1 derived ECM

    Surface immobilization of ECMs and their subsequent characterization have been described in detail elsewhere. Briefly, SCP-1 cells were seeded at a density of 1×104 cells per cm² on poly-(octadecene-alt-maleic anhydride) (POMA) and human fibronectin (FN; 5 µg/cm²) coated glass slide (Fig. 1A). Cultures were decellularized at day 10 using warm double distilled water supplemented with 20 mM ammonium hydroxide (Sigma) and ECMs were obtained by gentle agitation for 10 min at room temperature.

    CD34+ hematopoietic stem and progenitor cell purification and ECM culture

    G-CSF (granulocyte colony-stimulating factor) mobilized peripheral blood was obtained from healthy donors after informed consent (ethical approval no. EK221102004, EK47022007; the Ethics Committee of the Technische Universität Dresden). CD34+ HSPCs were purified using CD34 antibody-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec). CD34+ HSPCs were plated in 6 well plates with (ECM) or w/o (PCD) ECM scaffolds in CellGro medium (CellGenix) supplemented with 2.5 ng/ml SCF, IL-3 and Flt-3 each (all Miltenyi Biotec) for 5 days without medium change.

    Flow cytometry and fluorescent activated cell sorting (FACS)

    Cells were harvested by three washes in PBS/ 5% FCS. Antibody staining was performed according to manufacturer's protocol: CD34-APC (Miltenyi Biotec) CD45-FITC (Immunotech), and CD61-PE (Becton Dickinson). For dead cell exclusion, 4′,6-Diamidin-2-phenylindol (DAPI) was used. Corresponding human immunoglobulin G controls were used. LSRII and FACSAria (both Becton Dickinson) were used for flow cytometry measurement and sorting, respectively (Fig. 1C). Analyses were done using FlowJo software version 7.6.5 (Tree Star).

    Colony forming unit cell assay (CFU-GEMM)

    To identify HSPC clonogenicity and myeloid differentiation capacity colony-forming unit cell assay was performed using 500 cells in 3 ml semisolid human StemMACS medium complete with Epo (Miltenyi Biotec). Colony forming unit erythrocyte (CFU-E) and myeloid white cell colonies, including CFU-granulocyte (CFU-G), CFU-granulocyte/macrophage (CFU-GM) and CFU-macrophage (CFU-M) were counted according to standard criteria.

  • Funding statement

    Financial support from the DFG SFB655 grant ‘From cells to tissues’ (subproject B2 to C.W. and M.B. and subproject B10 to T.C) and the DKMS´Mechthild Harf Research Grant´(DKMS-SLS-MHG-2016-02 to A.J) is gratefully acknowledged.

  • Acknowledgements

    The authors would like to thank Katrin Müller for technical support.

  • Ethics statement

    G-CSF (granulocyte colony-stimulating factor) mobilized peripheral blood was obtained from healthy donors after informed consent (ethical approval no. EK221102004, EK47022007; the Ethics Committee of the Technische Universität Dresden).

  • References
  • 1
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    2
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    3
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    4
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    5
    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum Lorem ipsum Lorem ipsum Lorem ipsum Lorem ipsum ipsum

    Lorem ipsum Lorem ipsum Lorem ipsum
    Matters12/20

    Cell-derived Bone marrow-mimetics induce erythroid lineage commitment of hematopoietic stem and progenitor cells ex-vivo

    Affiliation listing not available.
    Martin Kräter
    C

    Medical Clinic I, University Hospital Carl Gustav Carus Dresden

    [email protected]
    Angela Jacobi
    Biotechnology Center, Technische Universität Dresden
    Stefanie Tietze
    Biotechnology Center, Technische Universität Dresden
    Manja Wobus
    Medical Clinic I, University Hospital Carl Gustav Carus Dresden
    Carsten Werner
    Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials
    Triantafyllos Chavakis
    Institute for Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden
    Martin Bornhauser
    C

    Medical Clinic I, University Hospital Carl Gustav Carus Dresden

    [email protected]