title: Engineered insulin secretion from neuroendocrine cells isolated from human thyroid.
abstract: BACKGROUND
  Insulin-secreting beta-like cells are vulnerable to diabetic autoimmunity. We hypothesized that human thyroid neuroendocrine (NE) cells could be engineered to secrete human insulin, be glucose-responsive, and avoid autoimmunity.
  
  
  METHODS
  Collagenase-digested thyroid tissue was cultured and subjected to size-based fluorescence-activated cell sorting. Insulin secretion and storage in NE cells transduced with viral vectors carrying an insulin sequence was assessed by enzyme-linked immunosorbent assay (ELISA) and immunogold transmission electron microscopy (TEM). Baseline mRNA expression was assessed by Illumina expression array analysis. Transduction with retrovirus expressing transcription factors PDX1, NGN3, MAFA, or HNF6 altered mRNA expression in a custom polymerase chain reaction (PCR) array. Gastrin-releasing peptide (GRP) in conditioned medium and cell lysates was determined by reverse transcription (RT)-PCR, ELISA, and immunohistochemistry.
  
  
  RESULTS
  Isolation yielded an average of 2.2 × 10(6) cells/g thyroid tissue, which stained for calcitonin/calcitonin gene-related protein, expressed genes consistent with NE origins, and secreted GRP. Transduced cells secreted 56 % and retained 48 % of total insulin produced. Immunogold TEM revealed insulin in secretory vesicles. PDX1, NGN3, and MAFA overexpression increased expression of genes typical for hepatocytes and beta cells. Overexpression of HNF6 also increased the message of genes critical for glucose sensing.
  
  
  CONCLUSIONS
  Human thyroid NE cells can produce human insulin, fractions of which are both secreted and retained in secretory granules. Overexpression of HNF6, PDX1, or NGN3 enhances expression of both hepatocyte and beta cell typical mRNAs, including the message of proteins critical for glucose sensing. These data suggest that reimplantation of engineered autologous NE cells may develop as a viable treatment for diabetes mellitus type 1.
authors.affiliation: Section Endocrinology & Metabolism, Atlanta VA Medical Center, Decatur, GA, USA, pthule@emory.edu.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Thulé
authors.forename: Dingwu
authors.initials: D
authors.lastname: Jia
authors.forename: Susan
authors.initials: S
authors.lastname: Safley
authors.forename: Kereen
authors.initials: K
authors.lastname: Gordon
authors.forename: Graham
authors.initials: G
authors.lastname: Barber
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.forename: Soumya
authors.initials: S
authors.lastname: Nalli
authors.forename: Muhittin
authors.initials: M
authors.lastname: Onderci
authors.forename: Jyotirmay
authors.initials: J
authors.lastname: Sharma
authors.forename: John
authors.initials: J
authors.lastname: Shires
authors.forename: Collin J
authors.initials: CJ
authors.lastname: Weber
mesh_terms: D057005:Bioengineering / Q000379:methods*; D002478:Cells, Cultured; D003922:Diabetes Mellitus, Type 1 / Q000188:drug therapy; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D051559:Hepatocyte Nuclear Factor 6 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology* / Q000627:therapeutic use; D050417:Insulin-Secreting Cells / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D055099:Neuroendocrine Cells / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism
chemical_list: D051559:Hepatocyte Nuclear Factor 6; D007328:Insulin; D012333:RNA, Messenger