Allogeneic Related HLA Mismatch Mesenchymal Stem Cells for the Treatment of Engraftment Failure Following Autologous Hematopoietic Stem Cell Transplantation.

Primary graft failure is usually associated with a high mortality rate despite infusion of back up graft and haematopoietic growth factors. In animal models mesenchymal stem cells (MSC) stimulate haematopoiesis recovery after TBI and enhance engraftment of haematopoietic stem cells (Almeida-Porada et al, Exp hematol 1999). Co-infusion of autologous blood stem cells and MSC in cancer patients receiving high dose chemotherapy speed up haematopoietic recovery (Koç et al, JCO 2000). We have previously shown that MSC can engraft and improve the bone marrow microenvironment in a patient with end stage severe aplastic anaemia (Fouillard et al, Leukemia 2003).

We report a patient treated with MSC for aplastic anemia secondary to engraftment failure. A 40 year old nulliparous woman with acute myeloid leukaemia received an autologous bone marrow transplantation; conditioning regimen combined a 12 Gray TBI and cyclophosphamide (120 mg/kg). Primary graft failure occurred and persited despite back up marrow infusion. Partial recovery on polymorphonuclear (PMN) and haemoglobin (Hb) was obtained with granulocyte colony stimulating factor (G-CSF) and EPO. Thrombocytopenia remained below 50x10⁹/l. No residual leukaemic cells were detected

Three years after ABMT, allogeneic MSC were infused at a dose of 2,78x10⁶/kg. MSC were isolated from a HLA mismatched brother bone marrow (Osiris Therapeutics Inc Baltimore, MD). At time of MSC infusion, the marrow aspirate was hypocellular with no leukaemic blast cells. Blood cell counts were: PMN: 0.8x10⁹/l, platelets: 45x10⁹/l and Hb: 10.5 g/dl. No conditioning regimen and no prophylaxis of GVHD was given. Growth factors were discontinued.

After MSC infusion, a rapid haematopoietic recovery was observed on both PMN and platelet which reached a normal level. With a follow up of 18 months, the patient is alive and well.

Recovery of haematopoiesis was corroborated by an improvement of in vitro haematopoietic and stromal clonogenic assays. CFU-GM and CFU-F studied the day before MSC infusion, one month and one year after MSC infusion, increased strikingly (p<0.05). LTC-IC increased significantly one year after MSC infusion (p<0.05). There was no change in BFUE.

To further characterize the effects seen on haematopoiesis, we utilized a custom RayBiotech antibody array and compared proteins secreted by MSC of recipient before and one year after infusion. This array evidenced an increased secretion of proteins implied in haematopoiesis (Flt3l, GM-CSF, G-CSF, IL1, IL6, TPO, SDF1) one year after MSC infusion. Real time PCR confirmed an up-regulation of gene expression for GCSF, GM-CSF, IL1, IL6.

We studied MSC engraftment. We analysed the bone marrow biopsy extracted DNA for mesenchymal chimerism before MSC infusion, one month and one year post MSC infusion by real time quantitative PCR of the Y specific SRY gene: male DNA was not detected before infusion; a level of male DNA of 1/10⁵ was detected one month after MSC infusion.

This observation shows that MSC can induce haematopoietic tissue repair. MSC should be considered in the treatment of engraftment failure and other bone marrow failure states including severe idiopathic aplastic anaemia and accidental irradiation.