Murine allogeneic in vivo stem cell homing

Department of Research, Roger Williams Medical Center, Providence, Rhode Island, USA.
Journal of Cellular Physiology (Impact Factor: 3.84). 06/2007; 211(2):386-91. DOI: 10.1002/jcp.20945
Source: PubMed


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.

Download full-text


Available from: Jan Cerny,
  • Source
    • "Among the cellular changes, the important functional impairment seen is the reduced/defective homing ability of these cells in culture thereby hampering their transplantation potential. Homing is an important prerequisite for engraftment, which utilizes the ability of HSPCs to migrate, adhere and lodge within the bone marrow niches and involve the synergistic action of adhesion molecules and integrins [1]–[3]. Previous studies revealed that the defective homing observed in cultured cells is due to the down regulation of beta integrins and chemokine receptors especially CXCR4, which is an indispensible homing molecule crucial for the trafficking of stem cells into the bone marrow following intravenous infusion [4], [5]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. CB derived CD34(+) cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice. Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant procedures.
    PLoS ONE 01/2012; 7(1):e29383. DOI:10.1371/journal.pone.0029383 · 3.23 Impact Factor
  • Source
    • "Notably, the one-cell dose infusion protocol led to a fraction of engrafted recipients irrespective of both irradiation levels, number of cells and infusion time. We then reasoned, based on the rapid clearance of infused BM cells in 1–3 h (Cerny et al, 2002; Colvin et al, 2007), that more than a one-cell dose could provide higher chimerism and, in particular, higher recipient engraftment efficiencies with the same levels of myelosuppression and of cells infused. Lastly, to evaluate the final parameter regarding the number of infused subdoses, we infused the same number of cells in two, three or four equal subdoses within 28 h of myelosuppression (Table VB). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Minimal criteria requirements of stem cell replacement, conditioning regimen and modalities of infusion essential for cure of sickle cell disease (SCD) by bone marrow(BM)/stem cell transplantation or gene therapy must be established prior to clinical trials. The threshold of normal BM/stem cells for therapeutic correction of this red blood cell disorder was evaluated in the SAD murine SCD model from peripheral donor white blood cells. From 11 groups of stable chimeric SAD mice (5-92%) analyzed over approximately 2 years, mice with approximately 16% normal donor stem cells showed improvement of haematological and erythroid responses. Mice in the 26% chimeric group and above demonstrated substantial amelioration of organ pathologies with generalized decreased iron deposits, fibrosis and reached normal lifespan. Subsequently, the minimal myelosuppression concurrently with number and timing of infusions and number of BM cells was determined to reach therapeutic threshold in SAD mice. Higher myelosuppression (2 Gy vs. 1 Gy) and cell number in single infusion led to increased chimerism. Importantly, administration of three-equivalent cell subdoses within 28 h of mild myelosuppression resulted in 100% recipient engraftment at therapeutic levels. These studies established the long-term therapeutic chimeric threshold of normal white blood cells at approximately 26% and determined the minimal fractionated BM/stem cell doses concomitant with mild myelosuppression for significant correction of SCD in SAD mice.
    British Journal of Haematology 11/2009; 148(4):646-58. DOI:10.1111/j.1365-2141.2009.07985.x · 4.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell tracking in vivo continues to provide significant insights into hematopoietic cell function and donor cell engraftment after transplantation. The combination of proliferation tracking dyes and induced expression of reporters with advanced imaging modalities has led to better understanding of qualitative and quantitative aspects of hematopoietic cells' homing, seeding and engraftment. Currently, there is no single technique that allows in vivo tracking of cells with molecular resolution, thus several techniques need to be combined. Recent developments promise better implementation of non-invasive imaging modalities to study functional and molecular characteristics of stem cells.
    Immunological Investigations 02/2007; 36(5-6):713-38. DOI:10.1080/08820130701715803 · 1.99 Impact Factor
Show more