Generation of human iPSCs from fetal prostate fibroblasts HPrF.

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.

Dendritic Cell-Based Immunotherapy in Advanced Sarcoma and Neuroblastoma Pediatric Patients: Anti-cancer Treatment Preceding Monocyte Harvest Impairs the Immunostimulatory and Antigen-Presenting Behavior of DCs and Manufacturing Process Outcome.

Despite efforts to develop novel treatment strategies, refractory and relapsing sarcoma, and high-risk neuroblastoma continue to have poor prognoses and limited overall survival. Monocyte-derived dendritic cell (DC)-based anti-cancer immunotherapy represents a promising treatment modality in these neoplasias. A DC-based anti-cancer vaccine was evaluated for safety in an academic phase-I/II clinical trial for children, adolescents, and young adults with progressive, recurrent, or primarily metastatic high-risk tumors, mainly sarcomas and neuroblastomas. The DC vaccine was loaded with self-tumor antigens obtained from patient tumor tissue. DC vaccine quality was assessed in terms of DC yield, viability, immunophenotype, production of IL-12 and IL-10, and stimulation of allogenic donor T-cells and autologous T-cells in allo-MLR and auto-MLR, respectively. Here, we show that the outcome of the manufacture of DC-based vaccine is highly variable in terms of both DC yield and DC immunostimulatory properties. In 30% of cases, manufacturing resulted in a product that failed to meet medicinal product specifications and therefore was not released for administration to a patient. Focusing on the isolation of monocytes and the pharmacotherapy preceding monocyte harvest, we show that isolation of monocytes by elutriation is not superior to adherence on plastic in terms of DC yield, viability, or immunostimulatory capacity. Trial patients having undergone monocyte-interfering pharmacotherapy prior to monocyte harvest was associated with an impaired DC-based immunotherapy product outcome. Certain combinations of anti-cancer treatment resulted in a similar pattern of inadequate DC parameters, namely, a combination of temozolomide with irinotecan was associated with DCs showing poor maturation and decreased immunostimulatory features, and a combination of pazopanib, topotecan, and MTD-based cyclophosphamide was associated with poor monocyte differentiation and decreased DC immunostimulatory parameters. Searching for a surrogate marker predicting an adverse outcome of DC manufacture in the peripheral blood complete blood count prior to monocyte harvest, we observed an association between an increased number of immature granulocytes in peripheral blood and decreased potency of the DC-based product as quantified by allo-MLR. We conclude that the DC-manufacturing yield and the immunostimulatory quality of anti-cancer DC-based vaccines generated from the monocytes of patients were not influenced by the monocyte isolation modality but were detrimentally affected by the specific combination of anti-cancer agents used prior to monocyte harvest.

Intact Cell Mass Spectrometry as a Quality Control Tool for Revealing Minute Phenotypic Changes of Cultured Human Embryonic Stem Cells.

The stability of in vitro cell cultures is an important issue for any clinical, bio-industrial, or pharmacological use. Embryonic stem cells are pluripotent; consequently, they possess the ability to differentiate into all three germ layers and are inherently prone to respond to differentiation stimuli. However, long-term culture inevitably yields clones that are best adapted to the culture conditions, passaging regimes, or differentiation sensitivity. This cellular plasticity is a major obstacle in the development of bio-industrial or clinical-grade cultures. At present, the quality control of cell cultures is limited by the lack of reliable (epi)genetic or molecular markers or by the focus on a particular type of instability such as karyotype abnormalities or adverse phenotypic traits. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming cell status and for revealing potential divergences from the optimal state. We modeled both intrinsic and extrinsic changes in human embryonic stem cell (hESC) states using different experimental strategies and addressed the changes in cell status by intact cell mass spectrometry fingerprinting. The analysis of spectral fingerprints by methods routinely used in analytical chemistry clearly distinguished the morphologically and biochemically similar populations of hESCs and provided a biomarker-independent tool for the quality control of cell culture. Stem Cells Translational Medicine 2018;7:109-114.

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A.

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies.

Soluble Cripto-1 Induces Accumulation of Supernumerary Centrosomes and Formation of Aberrant Mitoses in Human Embryonic Stem Cells.

Chromosomal instability evoked by abnormalities in centrosome numbers has been traditionally considered as a hallmark of aberrant, typically cancerous or senescent cells. We have reported previously that pristine human embryonic stem cells (hESC) suffer from high frequency of supernumerary centrosomes and hence may be prone to undergo abnormal mitotic divisions. We have also unraveled that this phenomenon of multicentrosomal mitoses vanishes with prolonged time in culture and with initiation of differentiation, and it is strongly affected by the culture substratum. In this study, we report for the first time that Cripto-1 protein (teratocarcinoma-derived growth factor 1, epidermal growth factor-Cripto/FRL-1/Cryptic) produced by hESC represents a factor capable of inducing formation of supernumerary centrosomes in cultured hESC. Elimination of Cripto-1 signaling on the other hand restores the normal number of centrosomes in hESC. Linking the secretory phenotype of hESC to the centrosomal metabolism may help to develop better strategies for propagation of stable and safe bioindustrial and clinical grade cultures of hESC. From a broader point of view, it may lead to unravelling Cripto-1 as a micro-environmental factor contributing to adverse cell behaviors in vivo.