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Comparative Study
. 2007 May;211(2):386-91.
doi: 10.1002/jcp.20945.

Murine allogeneic in vivo stem cell homing(,)

Gerald A Colvin  1 , Jean-Francois Lambert, Mark S Dooner, Jan Cerny, Peter J Quesenberry
Affiliations
Comparative Study

Murine allogeneic in vivo stem cell homing(,)

Gerald A Colvin et al. J Cell Physiol. 2007 May.

Abstract

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2-mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin-Sca-1+ cells were labeled with CFSE and injected in non-myeloablated BALB/c mice. Fluorescent cell detection was via high-speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony-forming cell (HPP-CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP-CFC. Combining experiments and normalizing the 1-h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 +/- 29%, 72 +/- 21%, 84 +/- 35% of the 1 h level at 3, 6, and 24 h respectively. HPP-CFC assay showed no significant variation as a homing surrogate over 1-6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells.

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Figures

Fig. 1
Fig. 1
Surrogate transplant assay evaluation of allogeneic marrow homing. This shows the schema used for the surrogate assay.
Fig. 2
Fig. 2
Detection of CFSE fluorescent cells in blood (A) and whole bone marrow (B) harvested serially after transplant. One hundred five CFSE-stained Lin− Sca-1+ cells (C57BL/6J) were injected into unconditioned BALB/c. A MoFlo® high-speed sorter was used to analyze a minimum of 107 events per marrow sample. Fluorescent events were detected in bone marrow at 1, 3, and 6 h after infusion. The number of CFSE+ cells is shown per thousand blood cells or per million marrow cells analyzed. Bars show standard error of the mean (SEM). Number of animals per group is shown in white for bone marrow analysis.
Fig. 3
Fig. 3
Cellular decay assay of bone marrow cellularity after 10-Gy irradiation. Bone marrow was harvested from femurs and tibias 1 to 24 h after infusion of allogeneic marrow cells. The recipient mice were irradiated with 10-Gy 2 h prior to transplant, thus times indicated reflect the cellularity decay 3, 5, 8, and 26 h after irradiation. Based on the cellularity, an enrichment ratio was calculated; this ratio allows calculating the relative increase of non-irradiated allogeneic homed cells compared to the host bone marrow cells. Using this data, an enrichment ratio was used to correct progenitor assays and secondary engraftment data to allow quantization and comparison between groups (infravide). There were six groups of two mice pooled together; standard error of the mean is shown.
Fig. 4
Fig. 4
Surrogate evaluation of homing by high proliferative potential colony-forming cell (HPP-CFC) assay. At different times after transplantation of bone marrow cells into allogeneic radio-ablated recipients, marrow was collected and plated into two layer soft agar with seven cytokines in the lower layer. Colonies were counted according to size and density (see methods). The numbers of colonies per million cells plated are shown. Mean values (five plates per group of two recipients, three groups per time-point) and SEM are shown. The Kruskal–Wallis test was performed to compare all groups.
Fig. 5
Fig. 5
Secondary engraftment of bone marrow at different times after initial allogeneic homing. A: The percentage of chimerism at 8-weeks in bone marrow, spleen, and thymus by FACS staining for CD45.1 and CD45.2 is presented. The data was corrected according to the relative enrichment ratio observed after 10-Gy lethal irradiation. Mean and SEM is shown, there were respectively 13, 10, and 13 mice for each time point. The difference between 1 and 6 h were significantin all organs (P = 0.043, <0.001, and 0.001 for bone marrow, spleen, and thymus, respectively). A test for trend from 1 to 6 h was significant for spleen and thymus (P < 0.005). B: Serial analysis of the percent of relative secondary engraftment in three pooled experiments compared with 1 h homing values. Data were corrected for cellularity decay and normalized based on the average marrow chimerism of each experiment. This data shows that homing and the subsequent engraftment chimerism percent plateaus within 1 h. There was decreased homing and engraftment with marrow collected 6 h after primary homing before retrieval and secondary homing and engraftment. The difference between 1 and 6 h was significant (P = 0.017). A test for trend from 1 to 6 h was significant (P = 0.01). The number of animals in each time-point is seen in white on each data bar. The Kruskal–Wallis test was performed to compare all groups.
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