Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

Transplantation of GABAergic cells derived from bioreactor-expanded human neural precursor cells restores motor and cognitive behavioral deficits in a rodent model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder that is characterized by progressive dementia, choreiform involuntary movements, and emotional deterioration. Neuropathological features include the progressive degeneration of striatal γ-aminobutyric acid (GABA) neurons. New therapeutic approaches, such as the transplantation of human neural precursor cells (hNPCs) to replace damaged or degenerated cells, are currently being investigated. The aim of this study was to investigate the potential for utilizing telencephalic hNPCs expanded in suspension bioreactors for cell restorative therapy in a rodent model of HD. hNPCs were expanded in a hydrodynamically controlled and homogeneous environment under serum-free conditions. In vitro analysis revealed that the bioreactor-expanded telencephalic (BET)-hNPCs could be differentiated into a highly enriched population of GABAergic neurons. Behavioral assessments of unilateral striatal quinolinic acid-lesioned rodents revealed a significant improvement in motor and memory deficits following transplantation with GABAergic cells differentiated from BET-hNPCs. Immunohistochemical analysis revealed that transplanted BET-hNPCs retained a GABAergic neuronal phenotype without aberrant transdifferentiation or tumor formation, indicating that BET-hNPCs are a safe source of cells for transplantation. This preclinical study has important implications as the transplantation of GABAergic cells derived from predifferentiated BET-hNPCs may be a safe and feasible cell replacement strategy to promote behavioral recovery in HD.

New bioengineering insights into human neural precursor cell expansion in culture.

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.

A review of bioreactor protocols for human neural precursor cell expansion in preparation for clinical trials.

Tissue-specific human neural precursor cells (hNPCs) can be isolated from various regions of the developing or adult central nervous system and may serve as a viable source of cells in cell replacement therapies for the treatment of neurodegenerative disorders. However, in order for cell replacement strategies to become a routine therapeutic option for the treatment of neurodegenerative disorders, hNPCs should be generated under standardized and controlled conditions. Studies over the last two decades have focused on developing cell growth media and cell handling protocols for expansion and differentiation of hNPCs in culture. Key studies have reported the development of serum-free growth media and large-scale computer-controlled suspension bioreactors that can support high cell proliferation rates (doubling times < 3 days), multipotentiality, and potential neurogenic differentiation (more than 60% neurons). Moreover, bioengineering studies have focused on controlling culture conditions in suspension bioreactors including inoculation, hydrodynamics of culture, oxygen and nutrients transfer to the cells, monitoring in situ physiological parameters using process control techniques, and expansion for extended periods of time. In addition, in vitro and in vivo characterization of hNPCs have been performed, providing information on stem/progenitor cell characteristics, cell surface analysis, and appropriate type of cells to use in transplantation studies.

Bioreactor expansion of human neural precursor cells in serum-free media retains neurogenic potential.

Human neural precursor cells (hNPCs), harvested from somatic tissue and grown in vitro, may serve as a source of cells for cell replacement strategies aimed at treating neurodegenerative disorders such as Parkinson's disease (PD), Huntington's disease (HD), and intractable spinal cord pain. A crucial element in a robust clinical production method for hNPCs is a serum-free growth medium that can support the rapid expansion of cells while retaining their multipotency. Here, we report the development of a cell growth medium (PPRF-h2) for the expansion of hNPCs, achieving an overall cell-fold expansion of 10(13) over a period of 140 days in stationary culture which is significantly greater than other literature results. More importantly, hNPC expansion could be scaled-up from stationary culture to suspension bioreactors using this medium. Serial subculturing of the cells in suspension bioreactors resulted in an overall cell-fold expansion of 7.8 x 10(13) after 140 days. These expanded cells maintained their multipotency including the capacity to generate large numbers of neurons (about 60%). In view of our previous studies regarding successful transplantation of the bioreactor-expanded hNPCs in animal models of neurological disorders, these results have demonstrated that PPRF-h2 (containing dehydroepiandrosterone, basic fibroblast growth factor and human leukemia inhibitory factor) can successfully facilitate the production of large quantities of hNPCs with potential to be used in the treatment of neurodegenerative disorders.

Expansion of human neural precursor cells in large-scale bioreactors for the treatment of neurodegenerative disorders.

The transplantation of in vitro expanded human neural precursor cells (hNPCs) represents a potential new treatment alternative for individuals suffering from incurable neurodegenerative disorders such as Parkinson's disease (PD) and Huntington's disease (HD). However, in order for cell restorative therapy to have widespread therapeutic significance, it will be necessary to generate unlimited quantities of clinical grade hNPCs in a standardized method. We report here that we have developed a serum-free medium and scale-up protocols that allow for the generation of clinical quantities of human telencephalon-derived hNPCs in 500-mL computer-controlled suspension bioreactors. The average hNPC aggregate diameter in the bioreactors was maintained below a target value of 500 microm by controlling the liquid shear field. The human cells, which were inoculated at 10(5) cells/mL, exhibited a doubling time of 84 h, underwent a 36-fold expansion over the course of 18 days, and maintained an average viability of over 90%. The bioreactor-derived hNPCs retained their nestin expression following expansion and were able to differentiate into glial and neuronal phenotypes under defined conditions. It has also been demonstrated that these hNPCs differentiated to a GABAergic phenotype that has recently been shown to be able to restore functional behavior in rat models of HD and neuropathic pain (Mukhida, K. et al. Stem Cells 2007; DOI 10.1634/stemcells.2007-0326). This study demonstrates that clinical quantities of hNPCs can be successfully and reproducibly generated under standardized conditions in computer-controlled suspension bioreactors.

Survival, differentiation, and migration of bioreactor-expanded human neural precursor cells in a model of Parkinson disease in rats.

OBJECT: Fetal tissue transplantation for Parkinson disease (PD) has demonstrated promising results in experimental and clinical studies. However, the widespread clinical application of this therapeutic approach is limited by a lack of fetal tissue. Human neural precursor cells (HNPCs) are attractive candidates for transplantation because of their long-term proliferation activity. Furthermore, these cells can be reproducibly expanded in a standardized fashion in suspension bioreactors. In this study the authors sought to determine whether the survival, differentiation, and migration of HNPCs after transplantation depended on the region of precursor cell origin, intracerebral site of transplantation, and duration of their expansion. METHODS: Human neural precursor cells were isolated from the telencephalon, brainstem, ventral mesencephalon, and spinal cord of human fetuses 8-10 weeks of gestational age, and their differentiation potential characterized in vitro. After expansion in suspension bioreactors, the HNPCs were transplanted into the striatum and substantia nigra of parkinsonian rats. Histological analyses were performed 7 weeks posttransplantation. RESULTS: The HNPCs isolated from various regions of the neuraxis demonstrated diverse propensities to differentiate into astrocytes and neurons and could all successfully expand under standardized conditions in suspension bioreactors. At 7 weeks posttransplantation, survival and migration were significantly greater for HNPCs obtained from the more rostral brain regions. The HNPCs differentiated predominantly into astrocytes after transplantation into the striatum or substantia nigra regions, and thus no behavioral improvement was observed. CONCLUSIONS: Understanding the regional differences in HNPC properties is prerequisite to their application for PD cell restoration strategies.