Control of pelage hair follicle development and cycling by complex interactions between follistatin and activin

Members of the transforming growth factor β/bone morphogenetic protein (TGF‐β/BMP) family are involved in the control of hair follicle (HF) morphogenesis and cycling. The activities of several members of this family (activins and BMP‐2, ‐4, ‐7, and ‐11) are controlled by antagonists such as follistatin. Because follistatin‐deficient mice show abnormalities in vibrissae development, we explored the role of follistatin and activin in pelage HF development and cycling. We show here that during HF development follistatin mRNA was prominently expressed by hair matrix and outer root sheath keratinocytes as well as by interfollicular epidermal cells, whereas activin βA mRNA was mainly expressed in dermal papilla cells. Compared with age‐matched wild‐type controls, both follistatin knockout mice and activin βA transgenic mice showed a significant retardation of HF morphogenesis. Treatment of wild‐type embryonic skin explants with follistatin protein stimulated HF development. This effect was inhibited by addition of recombinant activin A protein. Activin βA transgenic mice demonstrated retardation of catagen entry, down‐regulation of BMP‐2, and up‐regulation of expression of its antagonist matrix GLA protein. These observations suggest that follistatin and activin interaction plays an important role in both HF development and cycling, possibly in part by regulating expression of BMP‐2 and its antagonist

Substantial Sex-Dependent Differences in the Response of Human Scalp Hair Follicles to Estrogen Stimulation In Vitro Advocate Gender-Tailored Management of Female Versus Male Pattern Balding

In this study, it was investigated how estrogens (17-β-estradiol, E2) affect the estrogen receptor (ER) expression and gene regulation of male versus female human scalp hair follicles in vitro. Anagen VI follicles from frontotemporal scalp skin were microdissected and organ-cultured for up to 9 d in the presence of E2 (1–100 nm). Immunohistochemistry was performed for ERβ-expression, known to be predominant in human scalp hair follicles, and for TGF-β2-expression (as negative key hair growth modulator), and E2-responsive genes in organ-cultured human scalp hair follicles (48 h, 10 nM) were explored by cDNA microarray, using a commercial skin focus chip (Memorec, Cologne, Germany). The distribution pattern of ERβ and TGF-β2-immunoreactivity differed between male and female hair follicles after 48 h culture. Of 1300 genes tested, several genes were regulated sex-dependent differently. The study reveals substantial sex-dependent differences in the response of frontotemporal human scalp hair follicles to E2. Recognition and systematic dissection of the E2-dependent gene regulation will be crucial for the development of more effective, gender-tailored management strategies for female versus male pattern balding

Development of an automated manufacturing process for large-scale production of autologous T cell therapies

Engineered T cell therapies have shown significant clinical success. However, current manufacturing capabilities present a challenge in bringing these therapies to patients. Furthermore, the cost of development and manufacturing is still extremely high due to complexity of the manufacturing process. Increased automation can improve quality and reproducibility while reducing costs through minimizing hands-on operator time, allowing parallel manufacture of multiple products, and reducing the complexity of technology transfer. In this article, we describe the results of a strategic alliance between GSK and Miltenyi Biotec to develop a closed, automated manufacturing process using the CliniMACS Prodigy for autologous T cell therapy products that can deliver a high number of cells suitable for treating solid tumor indications and compatible with cryopreserved apheresis and drug product. We demonstrate the ability of the T cell Transduction – Large Scale process to deliver a significantly higher cell number than the existing process, achieving 1.5 × 1010 cells after 12 days of expansion, without affecting other product attributes. We demonstrate successful technology transfer of this robust process into three manufacturing facilities

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials

Background: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. Minimizing the number of intermediate technical steps of cell sample preparation will increase reproducibility of results. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. Methods: Fresh whole blood samples were obtained from consented healthy donors preserved either in PAXgene Blood RNA tubes or used for cell sorting. Target cells were enriched using magnetic microbeads and an autoMACS Pro Separator (Miltenyi). Cells were enumerated prior to magnetic cell sorting using a Siemens ADVIA® 120 Hematology System. Flow cytometric analysis for purity was performed before and after the magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). Results: Positive magnetic-activated cell separation (MACS) coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, CD45+ pan leucocytes. RNA quality from enriched cells was above eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. Conclusion: The proposed workflow generates reproducible cell-type specific transcriptome data for CD14+ -, CD3+ -, CD4+ -, CD8+ -, CD15+ -, CD19+ -, CD56+ -, and CD45+- cells, which can be translated to clinical settings and used to generate clinically relevant gene expression biomarkers from whole blood samples. This procedure facilitates the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols