The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of MSC from mouse BM (mBM), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of MSC, we hypothesized that a potential reason for the low yield of mMSC from mBM is the flushing of the marrow used to remove single cells suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of MSC physically associated with the abluminal surface of blood vessels.

Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs that yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least two orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal cell populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single cells suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays.

Since the first CB transplant (CBT) was performed by Gluckman et al in 1988, >16,000 patients have received this procedure for a variety of malignant and non-malignant diseases. Despite the lower frequencies of graft versus host disease (GvHD) and event-free survival rates comparable to those achieved after unrelated allogeneic marrow transplantation, a major disadvantage of CBT is the low cell dose, which results in slower engraftment and an elevated risk of engraftment failure by comparison with marrow or PBPC transplantation, even following transplantation with two CB units. A significant shortcoming of the cytokine-driven suspension culture systems initially envisioned for ex vivo expansion of CB cells is their failure to incorporate cues provided by the stromal cell-mediated microenvironment of the bone marrow. Seeking to enhance the efficiency of these culture systems, we have therefore incorporated mesenchymal precursor cells (MPC), a key cellular constituent of the hematopoietic stem cell niche. Optimisation of the expansion culture system and validation of the expanded product in immunodeficient NOD/SCID/IL-2RγNull (NSG) mice led to a clinical trial using haploidentical MSC (from a family member) and more recently using immunoselected allogeneic MPC (Mesoblast Ltd). More than 30 patients with a range of hematological malignancies have now been transplanted with a combination of two CB units, one unmanipulated, the second expanded on MPC. In fulfillment of our hypothesis, a significant enhancement in the rapidity of neutrophil and platelet reconstitution was observed, derived from the expanded unit that was superseded by long-term engraftment from the unmanipulated unit.


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  • Received: 05 March 2012
  • Accepted: 28 March 2012
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