Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation

Replacement of a diseased organ with an autologously derived tissue is an ideal therapy for some medical problems. However, it is difficult to recreate many adult human tissues in vitro due to the functionally necessary architecture of most organs and the lack of understanding of methods to direct the development of the organ of interest. The parathyroid gland is ideal for in vitro organ development because this gland is relatively simple, is transplantable, and is commonly affected by a surgical complication rather than an autoimmune disease. We have investigated thymus as a source of autologous endoderm and parathyroid-like precursor cells. Human thymus cells were treated with a differentiation protocol we developed with human embryonic stem cells (The Bingham Protocol) that utilizes timed exposures to Activin A and soluble Sonic hedgehog (Shh). We incrementally changed the protocol to optimize the differentiation of the thymus cells into parathyroid-like cells. The final protocol used 50 ng/mL Activin A and 100 ng/mL Shh over 13 weeks. The differentiated cells expressed the parathyroid markers parathyroid hormone (PTH), calcium sensing receptor, chemokine receptor type-4 (CXCR4), and chorian-specific transcription factor (GCM2) as measured by reverse transcription-polymerase chain reaction and PTH enzyme-linked immunosorbent assay. Cultured thymus cells without Activin A or Shh exposure did not secrete PTH nor express similar markers. The differentiated cells released PTH, which was suppressed in response to increased calcium concentration. The chemically differentiated cells did not form tumors in immune-compromised mice. Our protocol recreated cells with markers of parathyroid tissue that responded as parathyroid cells to physiologic stimuli. This approach is a further step toward a strategy to restore parathyroid function using autologous cells that were directed to differentiate by nongenetic in vitro manipulation.