Factors influencing engraftment of porcine hematopoietic progenitor cells in non-human primates
Background Cytokine-mobilized frozen-thawed porcine peripheral blood progenitor cells (PBPC) survive <3 weeks in baboons preconditioned with a non-myeloablative regimen, which includes anti-thymocyte globulin (ATG) and mycophenolate mofetil (MMF). The aim of the present study was to identify factors that might compromise the development of mixed hematopoietic chimerism in baboons transplanted with porcine PBPC.
Materials and methods PBPC were cytokine-mobilized with pSCF, pIL3 and hG-CSF and collected by leukapheresis from miniature swine. Baboon bone marrow (BM) was obtained by needle aspiration from naïve baboons. The influence of cryopreservation on PBPC was tested in vitro by enumeration of colony-forming units (CFU) in methylcellulose and cobblestone area-forming cell (CAFC) subsets in stromal-associated long-term cultures on fresh and frozen-thawed PBPC. The effects of ATG (Upjohn) and MMF (Roche) on porcine PBPC and baboon BM were tested in vitro by adding varying doses of ATG and MMF to porcine and baboon CFU assays. One baboon was treated with increasing doses of MMF (100-500 mg/kg per day continuous i.v., no peak levels) and sequential BM aspirations were tested for CFU assays.
Results Cytokine-mobilized PBPC had similar numbers of progenitor cells (measured by CFU or CAFC assays), when compared to porcine BM. Freezing-thawing of two separate PBPC samples cryopreserved in 48 and 80 ml volumes had no effect on porcine CFUs, but reduced the recovery of CAFCs by >90%. While ATG had no effect on growth of porcine or baboon CFUs, MMF completely inhibited the development of porcine and baboon CFUs at a concentration of 1 μg/ml or higher. In vivo administration of MMF resulted in plasma mycophenolate mofetil acid levels of 10-30 mg/ml and was associated with decreased CFU numbers in the baboon BM.
Conclusions Cryopreservation potentially prevents engraftment of porcine PBPC by reducing the content of progenitor cells. MMF (but not ATG) also has a negative effect on porcine or baboon progenitor cells. Therefore, the use of fresh porcine PBPC and the omission of MMF (or the use of low-dose MMF) might improve the survival of porcine PBPC and the induction of mixed hematopoietic chimerism in baboons.