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. 2015 Jun 30;4(2):e1066949. doi: 10.1080/21659087.2015.1066949

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 6 (See previous page). — Haematopoietic progenitor cells are retained in split femur transplants although at a low frequency. (A) Cryosection demonstrating the population of the secondary ossification center (SOC) of a split femur with haematopoietic cells 14 d after transplantation (3 transplants). (B) Genetic tracing reveals the presence of host haematopoietic cells (Ly-EGFP⁺) in the bone marrow cavity of a transplanted split femur (Tie2-Cre;R26-RFP) 15 d after transplantation. MIP of a multi-photon image stack (2 transplants). (C) Cells from the grafted femur, flushed 14 d after transplantation, belong both to the donor (Tie2-Cre; R26-RFP) and to the host (Syk-Cre; R26-YFP) (3 pooled transplants). (D) Macrophages and neutrophils decrease most in the interval between transplantation and day 3, then maintain constant levels until the end of experimentation period. For this experiment cells recovered from 3 or 4 transplants per time point were pooled and stained. (E) Propidium iodide cell cycle analysis for haematopoietic cells recovered from transplant at several time points show an initial increase in proliferation potential until day 7, that decreases with time until the end of the observation time (n = 3 or 4 transplants per time point). (F) Transplanted split femurs retain progenitor cells with myeloid differentiation capacity. Bone marrow cells from split or whole femur transplants were recovered 14 days after transplantation and the myeloid differentiation potential was analyzed in semisolid methylcellulose cultures. Shown is the contribution of the indicated colony types in (%) of the total colony number recovered (100%). Ctrl denotes freshly recovered femoral BM, half transplantation of a split femur, whole transplantation of an intact femur. Days indicate time after seeding at which the identity of the colonies was identified. (control = 5; half split = 20; whole femur = 4) (G) Morphology in methylcellulose (top panels) and histological appearance after cytocentrifugation and May–Grünwald-Giemsa staining (bottom panels) of representative colonies grown from cells recovered from transplants. CFU-M: macrophage colony; CFU-G: granulocytic colony, CFU-Mix: multilineage colony.