Manufacturing autologous myoblast for regenerative medicine applications.

BACKGROUND: Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). METHODS: Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15-17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. RESULTS: 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the donor age may impact the differentiation ability of myoblasts. CONCLUSIONS: The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases.

Functional Myeloid-Derived Suppressor Cell Subsets Recover Rapidly after Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation.

Myeloid-derived suppressor cells (MDSCs) are regulatory cell populations that have the ability to suppress effector T cell responses and promote the development of regulatory T cells (Tregs). They are a heterogeneous population of immature myeloid progenitors that include monocytic and granulocytic subsets. We postulated that given the rapid expansion of myeloid cells post-transplant, these members of the innate immune system may be important contributors to the early immune environment post-transplant. To evaluate the kinetics of recovery and function of MDSCs after allogeneic hematopoietic stem cell transplant (HSCT), 26 patients undergoing allogeneic HSCT were studied at 6 time points in the first 3 months after HSCT. Both MDSC subsets recovered between 2 and 4 weeks, well before the recovery of T and B lymphocytes. MDSC subset recovery positively correlated with T, B, and/or double-negative T cell numbers after HSCT. MDSCs isolated from patients post-transplant were functional in that they suppressed third-party CD4(+) T cell proliferation and Th1 differentiation and promoted Treg development. In conclusion, functional MDSC are present early after HSCT and likely contribute to the regulatory cell population post-transplant.

Identification of the CD34 enumeration on the day before stem cell harvest that best predicts poor mobilization.

BACKGROUND: We questioned whether CD34 enumeration in peripheral blood on the day before planned collection would identify a patient population that could benefit from an augmented collection strategy during that mobilization attempt. STUDY DESIGN AND METHODS: A retrospective review of all adult patients who underwent a first mobilization attempt between January and December 2008 for autologous use was undertaken. Peripheral blood CD34 quantitation on the day before planned collection (Day -1) and day of planned collection (Day 0) was correlated with likelihood of a successful collection. RESULTS: Of 41 patients (15 with multiple myeloma, 20 with lymphoma, and six with other malignancies) who underwent mobilization 24 patients (58%) were harvested in 1 day (good mobilizers) with the remaining 17 patients (42%) either requiring more than 1 day to collect or were not collected (poor mobilizers [PMs]). A peripheral blood CD34+ count below 10 × 10(6) /L on Day -1 was optimal in identifying PMs (adjusted odds ratio of 7 [1.4-35]). Increasing the CD34 cutoff from 10 × 10(6) to 15 × 10(6) /L decreased the prediction of poor mobilization (positive likelihood ratio dropped from 3.3 to 2.2). CONCLUSION: Peripheral blood CD34 content of less than or equal to 10 × 10(6) CD34+ cells/L on the day before collection is predictive of poor mobilization whereas higher peripheral blood CD34 counts on Day -1 have a high likelihood of successful 1-day collection.

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy.

BACKGROUND AIMS: The ability of hematopoietic progenitor cells-apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented. METHODS: In this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2-7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage). RESULTS: The doses of nucleated and CD34(+) cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10(8) and 6.8 ± 4.3 × 10(6), respectively) and long- (4.0 ± 4.9 × 10(8) and 6.1 ± 3.4 × 10(6), respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10(9)/L (median 11 days for both short- and long-term storage) and platelets >20 × 10(9)/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10(6)/kg CD34(+) cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets). CONCLUSIONS: Cryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers.

BACKGROUND AIMS: Enumeration of viable CD34(+) cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34(+) cells/microL blood predicts the collection of at least 0.5x10(6) CD34(+) cells/kg patient weight. From the apheresis product, infusion of 2.5x10(6) viable CD34(+) cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/microL) by day 12-14 post-infusion. METHODS: We compared the CD34(+) cell numbers derived from Flow Count-based Stem-Kit; (Beckman Coulter) and Trucount tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur and BD FACSCanto cytometers on 12 granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples. RESULTS: Comparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/microL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R(2)>0.98. CONCLUSIONS: The two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.