Purpose-Due to the 4-6-month interval between a diagnostic amniocentesis and birth, clinical application of amniotic mesenchymal stem cell (AMSC)-based therapies demands a 3-stage cell manufacturing process, including isolation/primary expansion, cryopreservation, and thawing/ secondary expansion. We sought to determine the feasibility and cell yield of such a staged cell manufacturing process, within regulatory guidelines. Methods-HumanAMSCs isolated from diagnostic amniocentesis samples (n=11) were processed under FDA-accredited Good Manufacturing Practice. Expanded cells were characterized by flow cytometry and cryopreserved for 3-5 months. Cell release criteria included: >90% CD29+, CD73+, and CD44+; <5% CD34+ and CD45+; negative mycoplasma QPCR and endotoxin assay; and ≥70% viability.Results-Isolation and ample expansion of AMSCs was achieved in 54.5% (6/11) of the samples. Early processing and at least a 2mL sample were necessary for reliable cell manufacturing. Cell yield before cryopreservation was 223.2±65.4×10 6 cells (44.6-fold expansion), plus a 14.7×10 6 -cell backup, after 36.3±7.8 days. Cell viability post-thaw was 88%. Expanded cells maintained a multipotent mesenchymal progenitor profile.Conclusions-Human amniotic mesenchymal stem cells can be manufactured in large numbers from diagnostic amniocentesis, by an accredited staged processing, under definite procurement guidelines. These data further support the viability of clinical trials of amniotic mesenchymal stem cell-based therapies.

Summary:A 43-year-old woman with Philadelphia chromosome (Ph) positive chronic myelogenous leukemia in acute phase received high-dose chemotherapy followed by transfusion of 12 randomly selected units of umbilical cord blood. HLA analysis showed cells of one donor from day +10 to day +43 post-transfusion. This unit was HLA class II identical with that of the patient. Keywords: placental blood; unmatched; multiple donors; CML There is considerable interest in umbilical cord blood (UCB) transplants (for reviews see Refs 1-4). Most transplants have been from HLA-matched relatives. However, there is increasing use of HLA-matched unrelated donors (reviewed in Ref.3). In 1972, Ende et al 5 transfused UCB from eight non-HLA unrelated donors to a 16-year-old with ALL previously treated with conventional chemotherapy. Success was claimed based on showing RBCs from one donor for 8 months. Shen et al 6 described giving multiple units of HLA-unmatched UCB to four patients with solid tumors. A patient with CML in accelerated phase was transplanted with HLA-partially matched UCB from one donor by Laporte et al 7 who reported successful transplantation of the cord blood stem cells into a 26-year-old women. We report a patient who received 12 units of HLA-untyped UCB for CML in acute phase after high-dose chemotherapy. Case reportThe patient, a 43-year-old female, was diagnosed with Ph chromosome-positive CML in 1991. She was treated with hydroxyurea and interferon until 1993 when accelerated phase developed. She then received total body irradiation (TBI) and cyclophosphamide followed by an autotransplant. In April 1996 accelerated phase recurred. She was treated with cytarabine to which she responded briefly. Based on a previously approved Institutional Review Board (IRB) transplantation protocol involving unrelated UCB and after informed consent, the patient received busulfan (12 mg/kg) and cyclophosphamide (120 mg/kg) followed by infusion of 3.18 × 10 9 mononuclear cells obtained from UCB from 12 unrelated donors.On day +1 we began GVHD prophylaxis consisting of cyclosporine and solumedrol. Early signs of engraftment were observed on day +10 with islands of trilineage hematopoiesis noted in a bone marrow biopsy specimen. There were no clinical features of GVHD. Increased bilirubin developed between days +14 and +17 with a peak value at 7.6 mg/dl. The patient was discharged in a stable condition on day +21 with a WBC count of 2.6 × 10 9 /l. Fetal hemoglobinized RBC and RhD-positive RBC were detected in the blood between day +17 and day +21 (patient's blood type O RhD-negative). Southern blot analysis of DNA from blood cells on day +16 showed no bcr/abl rearrangement (previously detected). Growth factor therapy was stopped on day +31 when the WBC count was Ͼ10 × 10 9 /l. The WBC continued to rise and was 8.5 × 10 9 /l on day +53 with reappearance of Ph chromosome-positive cells. Hydroxyurea and interferon were begun on day +71 and she died on day +90 of leukemia. Umbilical cord blood collecting and processingUnits of UCB were ...

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