Faculty Bibliography

Learning Objective #1: Recognize methemoglobinemia & its sequelae Learning Objective #2: Recall medications that confer risk of iatrogenic methemoglobinemia & treatment CASE: An 89-year-old woman was admitted with abdominal discomfort plus five days of dysuria and urinary frequency. Previously, she had seen a urologist for chronic dysuria and was prescribed estrogen cream for vaginal atrophy. On admission, her vital signs were notable for oxygen saturation of 88%, unresponsive to oxygen supplementation. Other clinical findings included jaundice, right upper quadrant and suprapubic tenderness, anemia (hemoglobin 8 g/dL), serum creatinine 2. 2 mg/dL, unremarkable liver function testing and ultrasound imaging, plus a uri-nalysis consistent with a urinary tract infection (UTI). Her chest X-ray was unremarkable and an arterial blood gas (ABG) obtained off of oxygen demonstrated normal partial pressure of oxygen (PaO2). Given the discrepancy between oxygen saturation on pulse oximetry and PaO2, met-hemoglobinemia was suspected. Co-oximetry revealed oxyhemoglobin 70%, methemoglobin > 21%. The patient denied recent benzocaine, nitrates, or dapsone use, but eventually disclosed taking phenazopyridine for several weeks prior to admission. Intravenous methylene blue was administered, ceftriaxone was given for her UTI, and supportive care was provided until her clinical status returned to baseline. IMPACT/DISCUSSION: Methemoglobinemia, a condition whereby functional anemia and tissue hypoxia are precipitated by acutely elevated methemoglobin concentration, is often iatrogenic. Nonspecific symptoms plus anemia and acute kidney injury (AKI) can be mistaken for many ailments, highlighting the importance of thorough medication review and having a high index of suspicion when there is a recent use of medications such as oxidizing agents (e.g. topical anesthetics, dapsone), nitrates, phenytoin, or antimalar-ials. The pathophysiology of acute respiratory distress in methemo-globinemia relates to the reduced erythrocyte oxygen-carrying capacity of functionally impaired hemoglobin. Skin discoloration, he-molytic anemia, and AKI have also been associated with phenazopyridine toxicity. The pathophysiology of AKI is still being investigated but may be multifactorial, including renal tubular epithelial cell injury, heme pigment-induced nephropathy from hemo-lytic anemia, and hypoxic injury from methemoglobinemia itself.
Conclusion(s): Methemolglobinemia presents with non-specific symptoms (e.g. headache, dizziness, dyspnea, fatigue, mental status changes). When a patient exhibits hypoxia unresponsive to supplemental oxygen, chocolate-brown hued arterial blood, and a discrepancy between pulse oximetry and ABG, methemoglobinemia should be at the top of one's differential. History-taking should include a thorough medication review (including over-the-counter agents) and chemical exposures. Once the diagnosis is confirmed with co-oximetry, methylene blue and supplemental oxygen should be given when methemoglobin level is greater than 20%

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.