Abstract
Particularly within the last several years cord blood (CB) has become a well established hematopoietic source for unrelated transplantation in children. Assuming, however, a threshold of 2x107 nucleated cells (NC)/kg body weight required for transplantation in adults over 70kg, an average of only 25% of the CB units presently stored in the major CB banks contain sufficient NC to safely engraft patients. Thus far the majority of studies have assessed selective cytokine driven expansion of progenitors for transplantation, which however may be associated with exhaustion of stem cells and an increase of GvHD in adult patients. Therefore, we assessed cytokine production and hematopoiesis supporting stromal activity of CB derived unrestricted somatic stem cells (USSC, Koegler et al. JEM2004: 200: 2: 1–13) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines. Initiation of USSC generation was initiated from fresh (n=325) and cryopreserved CB (n=66). In the presence of H5100 medium/10−7M dexamethasone, USSC cultures from fresh CB were initiated from 57.1% of CB samples (n= 52 out of 91 CB samples), whereas in DMEM-medium/10−7M dexamethasone the frequency was 31.6% (n=74 out of 234 CB samples). The generation frequency from frozen HES separated CB was only 19.5%. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Luminex or ELISA analyses. To analyze hematopoiesis supporting activity, CB CD34+ cells were expanded in co-cultures with USSC, BMMSC or in the presence of Flt3-L, SCF, TPO. Expansion of CD34+ cells, total cells and colony-forming cells (CFC) were determined after 1, 2, 3 and 4 weeks of culture. USSC constitutively produced SCF, LIF, TGF-1ß, M-CSF, GM-CSF, VEGF, IL-1ß, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1a and HGF. When USSC were stimulated with IL-1ß, G-CSF was released. Production of SCF (p= 0.0104) and LIF (p= 0.046) was significantly higher in USSC compared to BMMSC. At 1, 2, 3 and 4 weeks, respectively co-cultivation of CD34+ cells on the USSC layer resulted in a 14.62±1.1-fold, 110.1±17.9-fold, 151.8±39.7-fold and 183.6±40.4-fold amplification of total cells and in a 30.56±4.4-fold, 101.4±27.5-fold, 64.67±15.8-fold and 29.4±3.1-fold amplification of CFC. Expansion achieved was significantly higher for USSC compared to BMMSC feeder layers. USSC produce functionally significant amounts of hematopoiesis supporting cytokines and are able to expand CD34+ cells from CB in significantly higher amounts compared to BMMSC. Therefore the USSC is a suitable candidate to direct ex-vivo expansion of hematopoietic CB cells for short term reconstitution.
Enhanced In Vivo Homing of Uncultured and Selectively Amplified Cord Blood CD34+Cells by Cotransplantation with Cord Blood-Derived Unrestricted Somatic Stem Cells