We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

We report a case of cushing's syndrome caused by ectopic adreocortical adenoma. The patient is a 37 years old woman, she was admitted to our hospital for " 2 years history of hypertension and weakness in both lower extremities for 2 months". Physical examination revealed: blood pressure 160/116 mmHg(1 mmHg＝0.133 kPa), body mass index 27.47 kg/m2, moon-face, increased fat in the neck and back, purple marks on abdominal skin, withⅡdegree edema of both lower extremities. Laboratory examination revealed that serum cortisol levels were elevated, loss of normal circadian rhythm, and serum adrenocorticotropic hormone (ACTH) was suppressed, the level of cortisol could not be suppressed in low dose desamethasone suppression test. Adrenal computed tomography ( CT) revealed a nodule in the right retroperitoneum, compression of the renal hilum, no bilateral adrenal adenoma and hyperplasia were found. This patient was diagnosed as corticotropin-independent Cushing's syndrome unequivocally. The clinical symptoms were relieved after successful laparoscopic retroperitoneum resection of the nodule. Pathological exam confirmed adrenocortical adenoma in ectopic adrenal tissue. Thus, we should consider the ectopic corticosteroid-secreting tumor in the context of corticotropin-independent Cushing's syndrome, especially when the imaging studies of adrenal revealed bilateral adrenal glands were normal or atrophic, which helped to make an appropriate strategy treatment.

Objective:To study the clinical manifestations, diagnosis and treatment of primary hyperparathyroidism (PHPT) in pregnancy.Methods:This study involved six pregnant patients with PHPT who were admitted to Ruijin Hospital of Shanghai Jiao Tong University from August 2014 to November 2019. Their clinical manifestations, treatment strategies (multidisciplinary consultation with departments including Obstetrics, Endocrinology, Neonatology, General Surgery, Anesthesiology and Intensive Care Unit), maternal complications and maternal and infant outcomes were described and retrospectively analyzed.Results:(1) The median age of the six patients was 34(23-38) years old. PHPT was diagnosed in one case before pregnancy, four in the second or third trimester and one after delivery. The main clinical manifestations of four cases were nausea, vomiting, anorexia and other non-specific symptoms. Anemia was the most common maternal complication (five cases). Other complications included hypercalcemia crisis with renal failure (one case), multiple bone destruction and osteoporosis (one case). The median levels of serum calcium, parathyroid hormone and 25-hydroxyvitamin D were 3.08 (2.84-4.21) mmol/L, 216.7(93.1-2 603.6) ng/L and 29.66 (13.50-90.24) nmol/L, respectively. Results of parathyroid ultrasonography showed all patients had hypoechoic focus. Four cases underwent routine parathyroid radionuclide imaging showing abnormal radioactive concentration areas. (2) Obstetricians initiated the multidisciplinary team (MDT) for all patients to determine the treatment plans after communication with the patients and their families. One case received surgical treatment five months after delivery. Three cases received surgical treatment in the second trimester and among them, one terminated the pregnancy in the second trimester and the other two continued and achieved good pregnancy outcomes. Two cases underwent surgical treatment after giving up pregnancy and induced abortion. Postoperative pathology revealed that five cases were isolated parathyroid adenoma and one was parathyroid carcinoma. All of the patients were followed up and no abnormal serum calcium was reported. (3) One newborn had hypocalcemic convulsions one month after birth and was recovered by intravenous calcium supplementation. The newborn was followed up and the serum calcium level was normal so far. The other two neonates had normal serum calcium during follow-ups, but one of them was diagnosed with autism at the age of four and is now undergoing rehabilitation treatment.Conclusions:Pregnancy complicated by PHPT may lead to serious maternal and infant complications. MDT consultation ensures timely diagnosis, comprehensive treatment for the patients and better pregnancy outcomes.