Choosing the source of healthy controls for studies on myeloid malignancies: all bone marrow cells are created equal, but some are more equal than others
Stem Cell Research & Therapy volume 14, Article number: 36 (2023)
Bone marrow samples from discarded femoral heads are often used as healthy controls in studies investigating the in vitro characteristics of cells from patients with hematologic malignancies. Since patient samples are usually derived from iliac crest aspirates, this carries the risk that the properties of the cells from both sources might be different due to the site and method of harvesting. Comparing BM cells from iliac crest aspirates and femoral heads from age-matched healthy donors, we show that, while mesenchymal stromal cells have indistinguishable properties between both sources, hematopoietic stem and progenitor cells (HSPC) from femoral heads show a considerable proliferative advantage in vitro. These data therefore suggest that experiments comparing leukemic cells from the iliac crest to healthy HSPC obtained from femoral heads should be interpreted with caution.
Femoral heads (FHs) from hip replacement surgery provide a rich source of healthy bone marrow (BM) that is often used as a control sample in experimental set-ups for BM obtained from patients with hematologic malignancies [1,2,3,4,5]. Although phenotypically identical, patient BM is usually obtained from iliac crest aspirates (ICAs). The assumption that the properties of ICA BM cells are similar to those from FHs carries the risk that data may be misinterpreted, in particular if the observed differences should actually be attributed to the site and method of BM harvest.
In young adult mice the site of BM does not influence the properties of hematopoietic stem and progenitor cells (HSPC) . However, with ageing, the majority of hematopoiesis shifts from the long bones to the pelvis, sternum, skull and vertebrae  with a concomitant increase in BM adiposity of the femur . This change in the composition of the BM microenvironment has a direct influence on the residing hematopoietic and mesenchymal cells , which could particularly be of concern for studies on acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) since both hip replacement surgery patients, AML and MDS patients are of advanced age and cells are obtained from either the femoral head or the pelvis, respectively. Furthermore, FH BM is kept in the bone prior to processing whereas BM aspirates from the iliac crest are stored in a syringe, tube or bag, which can affect the quality and properties of the cells .
Materials and methods
The materials and methods used in this study can be found in the additional file online.
In order to shed light on this issue, we performed functional analyses of BM samples from healthy donors < 60 years, obtained by either aspiration from the iliac crest or from femoral heads following hip replacement surgery. Using age-matched samples (Additional file 1: Fig. S1), we studied multiple in vitro characteristics of both HSPC and mesenchymal stromal cells (MSC) and indeed found significant differences in the properties of these cells. These findings could have important consequences for the interpretation of existing data and design of future studies using FH as BM controls.
We first analyzed the proportion of early hematopoietic progenitors in CD34+ cells isolated from ICA or FH BM by flow cytometry (Additional file 1: Fig. S2). Small differences in HSPC proportions could be found between different donors, but on average there were no significant differences between both sources (Fig. 1A, B), suggesting that neither the site of harvest nor the method of extraction of the BM affects the composition of the BM HSPC pool.
We next assessed the proliferation and differentiation capacity of HSPC, by performing proliferation cultures and colony-forming-unit (CFU) assays. After 7 days of suspension culture in serum-free medium supplemented with cytokines, we again could not detect any differences in the proportions of different progenitor populations between the cells from ICAs or FHs (Fig. 1C). However, expansion of HSPC from FHs was significantly higher compared to ICA HSPC, as shown by the total cell number after 7 days (Fig. 1D, 3.9 ± 0.6-fold expansion vs. 2.5 ± 0.4-fold expansion, respectively, p = 0.008). A similar observation was made after seeding CD34+ cells in methylcellulose. After 14 days of culture, HSPC from the femur formed a significantly higher number of colonies (Fig. 1E, 68.1 ± 16.7 colonies/1000 CD34+ cells from FHs vs. 39.0 ± 9.0 colonies for CD34+ cells from ICAs, p = 0.006). The number of all types of colonies was increased significantly for CD34+ HSPC from FHs (Fig. 1E), with the exception of the colony foming unit erythrocyte (CFU-E) due to a high variability between samples. Notably, regardless of clonogenic capacity, the proportion of granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM) colonies from FH CD34+ HSPC was almost threefold higher than from ICA CD34+ HSPC (Additional file 1: Fig S3, 4.3% ± 2.7% vs. 1.5% ± 1.5%, p = 0.02). Since GEMM colonies are known to originate from cells that are higher up in the hematopoietic stem cell hierarchy , this could suggest that CD34+ cells from FHs contain a higher number of primitive HSPC. However, this was not reflected by the immunophenotype, showing that CD34+ cells from ICAs had a slightly higher percentage of HSC than CD34+ cells from FHs, albeit not significant (Fig. 1B, 5.3% ± 3.7% vs. 3.0% ± 1.4%, p = 0.22). To investigate the stem cell potential of CD34+ cells, we performed long-term culture (LTC) assays, a surrogate in vitro assay for stem cell activity. After 6 weeks of culture on a stromal layer, followed by replating in methylcellulose, FH CD34+ HSPC generated a slightly higher number of colonies (Fig. 1F, 10.6 ± 0.7 colonies vs. 9.4 ± 1.2 colonies/1000 CD34+ cells for FHs and ICAs, respectively, p = 0.10), indicating an increase in more primitive HSPC with self-renewal properties. However, considering the limited size of this increase, this difference is not likely to affect comparative studies.
Finally, we compared the stromal compartment from FHs and ICAs BM samples, using MSC isolated from the mononuclear cell fraction by plastic adherence. MSC from both sources were negative for CD45 and CD34, and positive for the characteristic MSC markers CD90, CD105 and CD73 (Additional file 1: Fig. S4). Analysis of the proliferation capacity of MSC cultured from ICAs and FHs showed no significant differences in their cumulative population doubling (Fig. 2A). To determine whether MSC from either BM source had a higher tendency to display features of ageing, we performed senescence assays on cells of passage 6 using the β-galactosidase assay (Fig. 2B, C). A higher mean percentage of senescent cells for FH MSC (23.1% ± 18.2) was observed compared to ICA MSC (8.9% ± 11.0), but due to a high variability between samples from each group, this difference was not significant.
Adipogenic and osteogenic differentiation has been shown to be dysregulated in diseases, such as MDS and AML . We therefore analyzed the differentiation potential of MSC from ICAs and FHs into adipogenic and osteogenic lineages. Image-based quantification of the staining of lipid droplets and calcium deposits showed a similar differentiation potential into both lineages (Fig. 2D-F). Lastly, since MSC provide vital support for HSPC homeostasis in the BM, we examined the capacity of FH and ICA MSC to support HSPC culture. Flow cytometry analysis of HSPC after culture showed no differences in the proportion of HSPC subpopulations (Fig. 2G) or HSPC number (Fig. 2H), indicating that potential to provide the hematopoietic support of MSC from both BM sources is approximately equal.
In summary, while MSC from both ICAs and FHs seem to proliferate, differentiate and support HSPC in a similar manner, BM CD34+ cells from FHs expand at a higher rate and contain more CFU, in particular immature CFU, compared to BM CD34+ cells from ICAs. These differences may be explained by intrinsic factors (e.g., a higher proliferation rate or a less quiescent state of HSPC) but could also be caused by extrinsic factors (e.g., differences in storage and processing methods including mechanical stress). In the BM, HSPC reside in a hypoxic microenvironment and exposure to oxygen can induce a loss of stem cell potential , which is demonstrated by the fact that isolation of HSPC from BM or cord blood under hypoxic conditions increases the number of primitive HSPC . Furthermore, mechanical stress can induce differentiation and a loss of primitive HSPC (15). Indeed, since ICA HSPC are stored in a collection bag, it is likely that they are exposed to higher levels of oxygen and mechanical stress, while FH HSPC remain in their natural microenvironment until mononuclear cell isolation. We hypothesize that this exposure could explain the lower proliferation capacity of ICA HSPC.
In conclusion, although FHs remain an attractive source of healthy BM due to their widespread availability and relatively low cost, our in vitro results indicate that CD34+ cells from FHs are not entirely comparable to patient samples obtained from ICAs as they show higher proliferative capacity of HSPC. Thus, in vitro studies using CD34+ HSPC from FHs as healthy controls in experimental set-ups examining myeloid malignancies should be interpreted with caution. Moreover, in future it would be advisable to obtain healthy BM samples from sources that more closely resemble the harvesting and processing protocols of patient BM.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Access to data files and further information is available upon request by contacting Mark van der Garde, email@example.com, tel. + 49 89 4140 6318.
Acute myeloid leukemia
Colony-forming unit erythrocyte
Colony-forming unit granulocyte, erythrocyte, monocyte, megakaryocyte
Hematopoietic stem cell
Hematopoietic stem and progenitor cell
Iliac crest aspirate
Long-term cell culture
Mesenchymal stromal cell
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We would like to thank the staff and donors of the Stiftung Aktion Knochenmarkspende, Bayerische Stammzellbank, Gauting for providing us the healthy bone marrow aspirates and Dr. M. Nolde and Dr. D. Hausmann of the Sana Klinikum München for providing us with femoral heads.
Open Access funding enabled and organized by Projekt DEAL. This work was supported by the German Cancer Consortium joint funding program (DKTK CHOICE (MG and KSG) as well as the Deutsche Forschungsgemeinschaft (SFB 1243 A09, MCB and KSG) and the German Jose Carreras Leukämiestiftung (DJCLS R14/18, JR and KSG). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
All human materials used in this study was obtained after informed written consent by the donor and with approval of the ethics committee of the Technische Universität München. Project title: Analyse der klonalen Hierarchie in MDS und die Beteiligung der Nische an der Progression in eine sekundäre AML. Institutional approval committee: Ethics committee TUM, Prof. Dr. Georg Schmidt. Approval number: TUM 538/16. Date of approval: December 14, 2016.
The authors declare no competing interests or other disclosures.
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Rivière, J., Hock, J., Buck, M.C. et al. Choosing the source of healthy controls for studies on myeloid malignancies: all bone marrow cells are created equal, but some are more equal than others. Stem Cell Res Ther 14, 36 (2023). https://doi.org/10.1186/s13287-023-03257-z