The authors acknowledge the use of the ICP-MS facility within the UC Center for Environmental Implications of Nanotechnology in CNSI at UCLA for their assistance with optimizing the protocol for 58Fe measurements. The study was supported by the NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (K01DK127004, to VS) and NIH National Institute of Child Health and Human Development (NICHD) (R01HD096863, to EN).

All animal protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California Los Angeles.
1. Preparation of 58Fe-Tf
NOTE: The protocol uses 58Fe; however, an identical protocol can be used for 57Fe. Either isotope can be used and disposed of as a standard iron chemical without additional precautions.
<ol>
	<li>Dissolve 58Fe in 12 N HCl at 50 &#181;L of HCl/mg of 58Fe.
	<ol>
		<li>Add HCl to the metal in the glass vial supplied by the vendor, and replace the cap loosely. To dissolve the iron, warm the 58Fe/HCl solution to 60 &#176;C for 1 h. If still not dissolved, leave the solution overnight at room temperature in the fume hood to dissolve. 
		NOTE: Dissolved 58Fe/HCl solution is yellowish-orange in color. 
		Fe3O4(s) + 8HCl(aq) &#8594; Fe(II)Cl2(aq) + 2Fe(III)Cl3(aq) + 4H2O</li>
	</ol>
	</li>
	<li>Oxidize any remaining Fe(II)Cl2 to generate the Fe(III)Cl3 solution.
	<ol>
		<li>Warm up the 58Fe/HCl solution to 60 &#176;C with the cap off to facilitate oxidation.</li>
		<li>Add 1 &#181;L of 35% H2O2 per 50 &#181;L of 58Fe/HCl solution to further facilitate oxidation. 
		Fe(II)Cl2(aq) + O2 + 4HCl &#8594; 4Fe(III)Cl3(aq) + 2H2O</li>
	</ol>
	</li>
	<li>Prepare the ferric chloride (58Fe(III)Cl3) solution.
	<ol>
		<li>Leave the ferric chloride solution in the hood at 60 &#176;C with the cap off to evaporate the sample. 
		NOTE: Evaporation may take between one and several days.</li>
		<li>Reconstitute 58Fe(III)Cl3 to 100 mM with ultrapure H2O, and calculate the amount of ultrapure H2O required based on the initial metal weight used in step 1.1 (molecular weight of 58Fe(III)Cl3 is 162.2).</li>
	</ol>
	</li>
	<li>Prepare 58Fe(III)-nitrilotriacetate (NTA) by incubating 58Fe(III)Cl3 with NTA at a 1:5 molar ratio in the presence of 20 mM NaHCO3.
	<ol>
		<li>Prepare 500 mM NTA in 1 N NaOH.</li>
		<li>Prepare 5x transferrin-loading buffer (0.5 M HEPES, pH 7.5; 0.75 M NaCl).</li>
		<li>Prepare 1 M NaHCO3 in ultrapure H2O.</li>
		<li>To a 15 mL conical tube, add 150 &#181;L of 100 mM 58Fe(III)Cl3 solution (from step 1.3.2), 150 &#181;L of 500 mM NTA prepared in 1 N NaOH, 480 &#181;L of ultrapure H2O, 200 &#181;L of 5x transferrin loading buffer, and 20 &#181;L of 1 M NaHCO3 solution.</li>
		<li>Incubate the mixture for 5 min at room temperature.</li>
	</ol>
	</li>
	<li>Load apo-Tf with 58Fe(III)-NTA to form 58Fe-Tf. 
	NOTE: This protocol was adapted from McCarthy and Kosman28.
	<ol>
		<li>Dissolve 500 mg of apo-Tf in 4 mL of 1x Tf-loading buffer.</li>
		<li>To the 15 mL conical tube in step 1.4.4 containing 1 mL of the 58Fe(III)-NTA solution, add 4 mL of apo-Tf solution. 
		NOTE: This is a 3:1 molar ratio of 58Fe-NTA with apo-Tf. Each Tf contains 2 Fe binding sites; excess 58Fe-NTA was added to ensure that Tf was fully loaded.</li>
		<li>To allow maximal loading of 58Fe-NTA onto apo-Tf, check that the solution is at pH 7.5, and adjust the pH, if necessary, with NaHCO3 or HCl.</li>
		<li>Incubate for 2.5 h at room temperature.</li>
	</ol>
	</li>
	<li>Remove excess unbound 58Fe(III)-NTA and released NTA.
	<ol>
		<li>Transfer the 58Fe-Tf solution to a molecular weight cutoff column (30 kDa cutoff) and centrifuge at 2,500 &#215; g for 15 min at room temperature.</li>
		<li>Wash the column with 10 mL of 1x transferrin-loading buffer and centrifuge at 2,500 &#215; g for 15 min at room temperature. Repeat the wash and centrifugation, perform a saline wash with 10 mL of saline, and centrifuge at 2,500 &#215; g for 15 min at room temperature.</li>
	</ol>
	</li>
	<li>Calculate the concentration of 58Fe-Tf. 
	NOTE: Due to the addition of excess 58Fe in step 1.5.2, assume that all transferrin is diferric. As 500 mg of apo-Tf was used, ~500 mg 58Fe-Tf was produced in step 1.5.4.
	<ol>
		<li>Measure the volume recovered from centrifugation after the saline wash in step 1.6.2.</li>
		<li>Divide 500 mg by the volume recovered to determine the concentration (in mg/mL) of the 58Fe-Tf solution.</li>
	</ol>
	</li>
	<li>Sterilize the 58Fe-Tf solution using a 0.22 &#181;m syringe filter; store at 4 &#176;C until ready to use. 
	NOTE: 58Fe-Tf solution was used between 1 to 4 weeks post preparation.</li>
</ol>
2. Set up timed mouse pregnancies
<ol>
	<li>Use 6- to 8-week-old female mice. Place animals on a low-iron diet (4 ppm iron) or standard chow (185 ppm iron) for 2 weeks prior to mating and maintain animals on the respective diets throughout pregnancy.</li>
	<li>Option 01: Confirm pregnancy by weight gain at E7.5.
	<ol>
		<li>Set up multiple breeding cages. For each cage, combine 2 females with 1 male overnight; the following day when animals are separated is considered embryonic day (E)0.5. Weigh females at E7.5 to determine if pregnant. Mate males again with females that did not gain weight. 
		NOTE: In WT C57BL/6, a weight gain of 1 g at E7.5 is a good indicator of pregnancy. This method ensures that implantation occurred within a specific 16 h timeframe, allowing for synchronous treatment of all animals that became pregnant during the same mating period.</li>
	</ol>
	</li>
	<li>Option 02: Confirm pregnancy by plug checks.
	<ol>
		<li>Combine 2 females with 1 male and perform daily plug checks to determine if copulation has occurred. 
		NOTE: This method may result in staggered pregnancies, and the presence of a plug does not guarantee pregnancy.</li>
	</ol>
	</li>
</ol>
3. Administer 58Fe-Tf intravenously to E17.5 pregnant mice
<ol>
	<li>Prepare 58Fe-Tf from step 1.8 for injection.
	<ol>
		<li>Prepare 58Fe-Tf solution at 35 mg/mL in saline; inject 100 &#181;L per mouse.</li>
		<li>Fill an insulin syringe with 100 &#181;L of the 58Fe-Tf solution. 
		NOTE: Each dose contains 3.5 mg of human 58Fe-Tf (5 &#181;g of 58Fe).</li>
	</ol>
	</li>
	<li>Anesthetize a pregnant mouse using isoflurane.
	<ol>
		<li>Use an isoflurane regulator with a chamber.</li>
		<li>Use the following settings: 5% isoflurane, 2 L/mL of O2, 2 min.</li>
		<li>Confirm the mouse is anesthetized by looking for lack of response to a toe pinch.</li>
		<li>Apply eye lubricant to the surface of the eye and place the mouse on a heating pad.</li>
	</ol>
	</li>
	<li>Slowly and carefully inject the 58Fe-Tf solution into the retro-orbital sinus.</li>
	<li>Allow the mouse to recover from anesthesia; do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Six hours post injection, euthanize E17.5 pregnant females by isoflurane overdose.
	<ol>
		<li>Perform a cardiac puncture to exsanguinate the mouse as a form of secondary euthanasia.</li>
		<li>Pin the feet down with needles for stabilization.</li>
	</ol>
	</li>
	<li>Collect the placentae and embryo livers.
	<ol>
		<li>Using sterile forceps and dissection scissors, carefully remove the uterus from the pregnant mouse. Cut off a placental fetal-placental unit, which comprises a single fetus and placenta in the amniotic sac surrounded by a portion of the uterus.</li>
		<li>Carefully cut through the uterus and amniotic sac without disturbing the fetus and placenta.</li>
		<li>Peel back the amniotic sac and remove the fetus and placenta.</li>
		<li>Cut the umbilical cord.</li>
		<li>Blot the fetus and placenta on a clean task wipe to remove the excess amniotic fluid.</li>
		<li>Record the weights of the whole placentae.</li>
		<li>Cut each placenta in half with a razor blade, place each half in a 2.0 mL tube, and snap-freeze in liquid nitrogen. 
		NOTE: Because 58Fe does not require special handling precautions and disposal, one-half of the placentae can be used for 58Fe measurement and the other half for any other analyses, including quantitation of transferrin receptor (TFR1) and ferroportin (FPN) expression by western blotting and qPCR.</li>
		<li>To collect embryo livers, sacrifice the embryo: use a razor blade to rapidly decapitate the embryo. 
		NOTE: At E17.5, all embryos in the uterus must be euthanized individually, even if they are not used in the study.</li>
		<li>Pin down the embryo for stabilization, leaving the abdomen exposed.</li>
		<li>Using dissection scissors, make a small incision where the umbilical cord was attached, insert one end of the dissection scissors into the incision, and perform a median plane cut toward the coronal plane about &#188; inch. Then, perform transverse plane cuts to expose the fetal liver.</li>
		<li>Use forceps to remove the fetal liver.</li>
		<li>Record the weights of the whole embryo livers.</li>
		<li>Place the whole embryo livers in 2 mL tubes and snap-freeze them in liquid nitrogen. 
		NOTE: Alternatively, only a portion of the embryo liver can be used for 58Fe measurement if additional analyses are desired. Using 2.0 mL tubes allows for better tissue homogenization than 1.5 mL tubes.</li>
	</ol>
	</li>
	<li>Store the tissues indefinitely at -80 &#176;C.</li>
</ol>
4. Process tissues for quantitative iron analysis by ICP-MS
<ol>
	<li>Process the placentae and fetal livers for the quantitation of nonheme iron.
	<ol>
		<li>Thaw placental halves and whole fetal livers, and weigh placental halves (see step 3.6.12 for recording fetal liver weights).</li>
		<li>Add 400 &#181;L of protein precipitation solution (0.53 N HCl, 5.3% TCA).</li>
		<li>Homogenize the tissue using an electric homogenizer.</li>
		<li>Incubate the samples at 100 &#176;C for 1 h.</li>
		<li>Cool the samples in room temperature water for 2 min.</li>
		<li>Open the caps to release pressure, then close the tubes again.</li>
		<li>Centrifuge at 17,000 &#215; g for 10 min at room temperature to pellet tissue debris.</li>
		<li>Carefully transfer the supernatant to a new labeled tube.</li>
		<li>Send samples off for ICP-MS analysis.</li>
	</ol>
	</li>
	<li>Process the placentae and fetal livers for the quantitation of heme-iron. 
	NOTE: Following extraction of nonheme iron in step 1, the iron remaining in the pellet is predominantly heme.
	<ol>
		<li>Record the weight of each pellet from step 4.1.7.</li>
		<li>Digest the pellets in 10 mL of concentrated 70% HNO3 supplemented with 1 mL of 30% H2O2 
		NOTE: Consult with the ICP-MS core or center to optimize the volume of HNO3 for specific studies; the volume will partly be dependent on sample weight.</li>
		<li>Heat the samples to 200 &#176;C for 15 min.</li>
		<li>Send the samples off for ICP-MS analysis. 
		NOTE: If distinguishing between heme and nonheme iron sources is not required and only total iron is measured, whole tissue can be digested in HNO3 as the first step.</li>
	</ol>
	</li>
</ol>
5. Data analysis
NOTE: Data from ICP-MS has been provided as 56Fe and 58Fe concentrations in ng/mL or mg, ppb (Table 1). 56Fe is the most abundant iron isotope in nature, and its measurement reflects iron accumulation in the placenta/embryo over the entire pregnancy, whereas 58Fe measurement reflects iron that was transferred during 6 h after injection.
<ol>
	<li>Subtract the natural abundance of 58Fe (0.28% of total Fe) from the measured 58Fe values.</li>
	<li>Calculate total nonheme 58Fe.
	<ol>
		<li>Calculate embryo liver total nonheme iron (ng) by first multiplying the iron concentration (ng/mL) calculated in step 5.1 by the volume (mL) during initial processing in step 4.1.2 to estimate total 58Fe.</li>
		<li>Calculate the amount of iron in the whole placenta by taking the total weight of the placenta measured in step 3.6.6 and dividing it by the weight of the placenta processed in step 4.1.1. Multiply this value by the total nonheme iron (ng) calculated in step 5.2.1 to obtain the total nonheme 58Fe content of the placenta.</li>
	</ol>
	</li>
	<li>Calculate total heme 58Fe.
	<ol>
		<li>Calculate total heme 58Fe by first multiplying the iron concentration (ng/mg) calculated in step 5.1 by the weight of the pellet (in mg) measured in step 4.2.1.</li>
		<li>Then, divide the total weight of the placenta measured in step 3.5.1 by the weight of the placenta pellet measured in step 4.2.1. Multiply this value by the total heme iron (ng) calculated in step 5.3.1 to obtain total heme 58Fe content of the placenta.</li>
	</ol>
	</li>
	<li>Sum the calculated nonheme and heme 58Fe values to determine the total iron content for each tissue.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63378/63378fig01.jpg" /> 
Figure 1: Visual summary of steps in the protocol. (A) Preparation of 58Fe-transferrin. (B) In vivo administration of 58Fe-transferrin. (C) Tissue collection and storage. (D) Processing of the placenta and embryo liver for quantitation of metal species by ICP-MS. Abbreviations: Fe = iron; NTA = nitrilotriacetic acid; Tf = transferrin; PPS = protein precipitation solution; Sup = supernatant; TCA = trichloroacetic acid; ICP-MS = inductively coupled plasma mass spectrometry. <a href="https://www.jove.com/files/ftp_upload/63378/63378fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Ganz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+iron+homeostasis.">Systemic iron homeostasis.</a> Physiological Reviews. 93 (4), 1721-1741 (2013).</li><li>Aschemeyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+ferroportin+defines+the+binding+site+and+an+alternative+mechanism+of+action+of+hepcidin.">Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin.</a> Blood. 131 (8), 899-910 (2018).</li><li>Sangkhae, V., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+iron+homeostatic+hormone+hepcidin.">Regulation of the iron homeostatic hormone hepcidin.</a> Advances in Nutrition. 8 (1), 126-136 (2017).</li><li>Sangkhae, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+maternal+iron+status+on+placental+and+fetal+iron+homeostasis.">Effects of maternal iron status on placental and fetal iron homeostasis.</a> Journal of Clinical Investigation. 130 (2), 625-640 (2020).</li><li>Whittaker, P. G., Lind, T., Williams, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+during+normal+human+pregnancy:+a+study+using+stable+isotopes.">Iron absorption during normal human pregnancy: a study using stable isotopes.</a> British Journal of Nutrition. 65 (3), 457-463 (1991).</li><li>Whittaker, P. G., Barrett, J. F., Lind, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+erythrocyte+incorporation+of+absorbed+non-haem+iron+in+pregnant+women.">The erythrocyte incorporation of absorbed non-haem iron in pregnant women.</a> British Journal of Nutrition. 86 (3), 323-329 (2001).</li><li>O'Brien, K. O., Zavaleta, N., Abrams, S. A., Caulfield, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+iron+status+influences+iron+transfer+to+the+fetus+during+the+third+trimester+of+pregnancy.">Maternal iron status influences iron transfer to the fetus during the third trimester of pregnancy.</a> American Journal of Clinical Nutrition. 77 (4), 924-930 (2003).</li><li>Young, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+hepcidin+is+associated+with+placental+transfer+of+iron+derived+from+dietary+heme+and+nonheme+sources.">Maternal hepcidin is associated with placental transfer of iron derived from dietary heme and nonheme sources.</a> Journal of Nutrition. 142 (1), 33-39 (2012).</li><li>Delaney, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+during+pregnancy+is+underestimated+when+iron+utilization+by+the+placenta+and+fetus+is+ignored.">Iron absorption during pregnancy is underestimated when iron utilization by the placenta and fetus is ignored.</a> American Journal of Clinical Nutrition. 112 (3), 576-585 (2020).</li><li>Klatt, K. C., Smith, E. R., Barberio, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+more+stable+understanding+of+pregnancy+micronutrient+metabolism.">Toward a more stable understanding of pregnancy micronutrient metabolism.</a> American Journal of Physiology-Endocrinology Metabolism. 321 (2), 260-263 (2021).</li><li>Fisher, A. L., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+homeostasis+during+pregnancy.">Iron homeostasis during pregnancy.</a> American Journal of Clinical Nutrition. 106, 1567-1574 (2017).</li><li>van Santen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron+regulatory+hormone+hepcidin+is+decreased+in+pregnancy:+a+prospective+longitudinal+study.">The iron regulatory hormone hepcidin is decreased in pregnancy: a prospective longitudinal study.</a> Clinical Chemistry and Laboratory Medicine. 51 (7), 1395-1401 (2013).</li><li>Millard, K. N., Frazer, D. M., Wilkins, S. J., Anderson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+expression+of+intestinal+iron+transport+and+hepatic+regulatory+molecules+explain+the+enhanced+iron+absorption+associated+with+pregnancy+in+the+rat.">Changes in the expression of intestinal iron transport and hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat.</a> Gut. 53 (5), 655-660 (2004).</li><li>Bothwell, T. H., Pribilla, W. F., Mebust, W., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+metabolism+in+the+pregnant+rabbit;+iron+transport+across+the+placenta.">Iron metabolism in the pregnant rabbit; iron transport across the placenta.</a> American Journal of Physiology. 193 (3), 615-622 (1958).</li><li>Dyer, N. C., Brill, A. B., Raye, J., Gutberlet, R., Stahlman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal-fetal+exchange+of+59+Fe:+radiation+dosimetry+and+biokinetics+in+human+and+sheep+studies.">Maternal-fetal exchange of 59 Fe: radiation dosimetry and biokinetics in human and sheep studies.</a> Radiation Research. 53 (3), 488-495 (1973).</li><li>Contractor, S. F., Eaton, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+iron+transport+between+maternal+and+fetal+circulations+of+a+perfused+lobule+of+human+placenta.">Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta.</a> Cell Biochemistry &amp; Function. 4 (1), 69-74 (1986).</li><li>Baker, E., Morgan, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+transferrin+in+placental+iron+transfer+in+the+rabbit.">The role of transferrin in placental iron transfer in the rabbit.</a> Quartly Jounrnal of Experimental Physiolology and Cognate Medical Sciences. 54 (2), 173-186 (1969).</li><li>Fleming, R. E., Feng, Q., Britton, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+mouse+models+of+iron+homeostasis.">Knockout mouse models of iron homeostasis.</a> Annual Review of Nutrition. 31, 117-137 (2011).</li><li>Soares, M. J., Varberg, K. M., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemochorial+placentation:+development,+function,+and+adaptations.">Hemochorial placentation: development, function, and adaptations.</a> Biology of Reproduction. 99 (1), 196-211 (2018).</li><li>Jones, H. N., Powell, T. L., Jansson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+placental+nutrient+transport--a+review.">Regulation of placental nutrient transport--a review.</a> Placenta. 28 (8-9), 763-774 (2007).</li><li>Rossant, J., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+development:+lessons+from+mouse+mutants.">Placental development: lessons from mouse mutants.</a> Nature Reviews Genetics. 2 (7), 538-548 (2001).</li><li>Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., Hirano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glucose+transporter+GLUT1+in+the+rat+placental+barrier:+possible+role+of+GLUT1+and+the+gap+junction+in+the+transport+of+glucose+across+the+placental+barrier.">Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier.</a> Cell and Tissue Research. 276 (3), 411-418 (1994).</li><li>Shin, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+GLUT1+and+connexin+26+in+the+rat+placenta.">Immunolocalization of GLUT1 and connexin 26 in the rat placenta.</a> Cell and Tissue Research. 285 (1), 83-89 (1996).</li><li>Bastin, J., Drakesmith, H., Rees, M., Sargent, I., Townsend, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localisation+of+proteins+of+iron+metabolism+in+the+human+placenta+and+liver.">Localisation of proteins of iron metabolism in the human placenta and liver.</a> British Journal of Haematology. 134 (5), 532-543 (2006).</li><li>Klausner, R. D., Ashwell, G., van Renswoude, J., Harford, J. B., Bridges, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+apotransferrin+to+K562+cells:+explanation+of+the+transferrin+cycle.">Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (8), 2263-2266 (1983).</li><li>Tsunoo, H., Sussman, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+transferrin+binding+and+specificity+of+the+placental+transferrin+receptor.">Characterization of transferrin binding and specificity of the placental transferrin receptor.</a> Archives of Biochemistry and Biophysics. 225 (1), 42-54 (1983).</li><li>Sangkhae, V., Nemeth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+iron+transport:+The+mechanism+and+regulatory+circuits.">Placental iron transport: The mechanism and regulatory circuits.</a> Free Radical Biology and Medicine. 133, 254-261 (2019).</li><li>McCarthy, R. C., Kosman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+analysis+of+iron+accumulation+by+endothelial+cells+of+the+BBB.">Mechanistic analysis of iron accumulation by endothelial cells of the BBB.</a> Biometals. 25 (4), 665-675 (2012).</li><li>Donovan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iron+exporter+ferroportin/Slc40a1+is+essential+for+iron+homeostasis.">The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis.</a> Cell Metabolism. 1 (3), 191-200 (2005).</li><li>Stefanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+hepcidin+and+its+agonist+mediate+resistance+to+selected+infections+by+clearing+non-transferrin-bound+iron.">Endogenous hepcidin and its agonist mediate resistance to selected infections by clearing non-transferrin-bound iron.</a> Blood. 130 (3), 245-257 (2017).</li><li>Ramos, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+distinct+pathways+of+hepcidin+regulation+by+acute+and+chronic+iron+loading+in+mice.">Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice.</a> Hepatology. 53 (4), 1333-1341 (2011).</li><li>Kulandavelu, S., Qu, D., Adamson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+function+in+mice+during+normal+pregnancy+and+in+the+absence+of+endothelial+NO+synthase.">Cardiovascular function in mice during normal pregnancy and in the absence of endothelial NO synthase.</a> Hypertension. 47 (6), 1175-1182 (2006).</li><li>Lu, C. C., Matsumoto, N., Iijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+transfer+and+body+distribution+of+nickel+chloride+in+pregnant+mice.">Placental transfer and body distribution of nickel chloride in pregnant mice.</a> Toxicology and Applied Pharmacology. 59 (3), 409-413 (1981).</li><li>Gunshin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slc11a2+is+required+for+intestinal+iron+absorption+and+erythropoiesis+but+dispensable+in+placenta+and+liver.">Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver.</a> Journal of Clinical Investigation. 115 (5), 1258-1266 (2005).</li></ol>

EN is a scientific co-founder of Intrinsic LifeSciences and Silarus Pharma and a consultant for Protagonist, Vifor, RallyBio, Ionis, Shield Therapeutics, and Disc Medicine. VS declares no conflicts.

Iron is important for many biological processes, and its movement and distribution within the body are highly dynamic and regulated. Stable iron isotopes provide a consistent and convenient alternative to radioactive isotopes for the assessment of the dynamics of iron homeostasis. A critical step in the protocol is keeping track of all the tissue weights and volumes. Iron is an element and therefore cannot be synthesized nor broken down. Thus, if all weights and volumes are carefully logged, all the iron within the syste...

Iron is critical for various metabolic processes, including growth and development, energy production, and oxygen transport1. Maintenance of iron homeostasis is a dynamic, coordinated process. Iron is absorbed from food in the duodenum and transported around the body in the circulation bound to the iron transport protein transferrin (Tf). It is utilized by every cell for enzymatic processes, incorporated into hemoglobin in nascent erythrocytes, and recycled from aged erythrocytes by macrophages. Iron is stored in the liver when in excess and lost from the body through hemorrhage or cell sloughing. The amount of iron in circulation is the result of the balance between the consumption and the supply of iron, the latter being tightly regulated by the hepatic hormone hepcidin (HAMP), the central regulator of iron homeostasis1. Hepcidin functions to limit iron bioavailability in blood by occluding or inducing ubiquitination and degrading the iron exporter ferroportin (FPN)2. Reduction in functional FPN leads to decreased dietary iron absorption, iron sequestration in the liver, and decreased iron recycling from macrophages1.
Hepcidin is regulated by iron status, inflammation, erythropoietic drive, and pregnancy (reviewed in 3). Given that iron homeostasis is highly dynamic, it is important to understand and measure the total iron pool and iron distribution and turnover. Animal studies traditionally relied on radioactive iron isotopes, a highly sensitive yet burdensome approach to measure iron dynamics. However, in more recent studies, including the study presented here4, nonradioactive, stable iron isotopes (58Fe) are utilized to measure iron transport during pregnancy5,6,7,8,9. Stable isotopes are valuable tools for studying nutrient metabolism (reviewed in 10). The use of stable iron isotopes in human studies demonstrated that i) iron absorption increases toward the end of gestation5,6, ii) transfer of dietary iron to the fetus is dependent on maternal iron status7, iii) maternally ingested heme iron is more readily incorporated by the fetus than nonheme iron8, and iv) iron transfer to the fetus is negatively correlated with maternal hepcidin levels8,9. These experiments measured iron isotopes in sera or their incorporation into RBCs; however, measurement of iron incorporated into RBCs alone may underestimate true iron absorption9. In the current study, both heme and nonheme iron are measured in tissues.
During pregnancy, iron is required to support the expansion of maternal red blood cell volume and for transfer across the placenta to support the growth and development of the fetus11. Fetal iron endowment is wholly dependent on iron transport across the placenta. During human12 and rodent4,13 pregnancy, hepcidin levels dramatically decrease, increasing plasma iron availability for transfer to the fetus.
The fundamentals of placental iron transport were initially characterized in the 1950s-70s using radioactive tracers (59Fe and 55Fe). These studies determined that iron transport across the placenta is unidirectional14,15 and that diferric transferrin is a major source of iron for the placenta and fetus16,17. The current understanding of placental iron transport is more complete, although some key iron transporters and regulatory mechanisms remain unknown. Mouse models have been essential for understanding iron regulation and transport18 because the key transporters and mechanisms are remarkably similar. Both human and mouse placentae are hemochorial, that is, maternal blood is in direct contact with the fetal chorion19. However, there are some notable structural differences.
The syncytiotrophoblast is the placental cell layer that separates the maternal and fetal circulation and actively transports iron and other nutrients20. In humans, the syncytiotrophoblast is a single layer of fused cells. In contrast, the mouse placenta consists of two syncytiotrophoblast layers21, Syn-I and Syn-II. However, gap junctions at the interface of Syn-I and Syn-II allow the diffusion of nutrients between layers22,23. Thus, these layers function as a single syncytial layer similar to the human syncytiotrophoblast. Additional similarities and differences between human and mouse placentae are reviewed by Rossant and Cross21. Placental iron transport is triggered by the binding of iron-Tf from maternal blood to the transferrin receptor (TfR1) localized on the apical side of the syncytiotrophoblast24. This interaction induces iron-Tf/TfR1 internalization via clathrin-mediated endocytosis25. Iron is then released from Tf in the acidic endosome26, reduced to ferrous iron by an undetermined ferrireductase, and exported from the endosome to the cytoplasm by a yet-to-be determined transporter. How iron is chaperoned within the syncytiotrophoblast also remains to be described. Iron is eventually transported to the fetal side by the iron exporter, FPN, localized on the basal or fetal-facing surface of the syncytiotrophoblast (reviewed in27).
To understand how physiological and pathological regulation of TfR1, FPN, and hepcidin affects placental iron transport, stable iron isotopes were utilized to quantitate iron transport from the maternal circulation to the placenta and embryo in vivo4. This paper presents the methods for preparing and administering isotopic iron-transferrin to pregnant mice, processing of tissues for ICP-MS, and calculating iron concentrations in tissues. The use of stable iron isotopes in vivo can be adapted to investigate iron regulation and distribution in different animal models to investigate physiologic and pathologic iron regulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>58Fe-iron metal</td>
			<td>Trace Sciences International</td>
			<td>Fe-58</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-15 centrifugal filter, 30 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC903024</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL</td>
			<td>Millipore Sigma</td>
			<td>CLS430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>75002432</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, Sorvall Legend RT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet: iron-deficient (4 ppm iron)</td>
			<td>Envigo Teklad</td>
			<td>TD.80396</td>
			<td></td>
		</tr>
		<tr>
			<td>Diet: standard chow (185 ppm iron)</td>
			<td>PicoLab</td>
			<td>5053</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor with 30 mm cutting edge</td>
			<td>VWR</td>
			<td>25870-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 4-1/2 inch length</td>
			<td>McKesson</td>
			<td>157-469</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer, Bio-Gen PRO200</td>
			<td>PROScientific</td>
			<td>01-01200</td>
			<td></td>
		</tr>
		<tr>
			<td>Human apo-transferrin (apo-Tf)</td>
			<td>Celliance</td>
			<td>4452-01</td>
			<td>no longer available, alternative: Millipore 616419</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), 35 wt.% solution in water</td>
			<td>Cole-Parmer</td>
			<td>EW-88216-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes, BD Lo-Dose U-100</td>
			<td>Fisher Scientific</td>
			<td>14-826-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VETone</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizor</td>
			<td>Summit Anesthesia Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal heat block</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge tube with flat screw-cap</td>
			<td>VWR</td>
			<td>16466-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL low-retention</td>
			<td>Fisher Scientific</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL low-retention</td>
			<td>Fisher Scientific</td>
			<td>02-681-321</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit, 0.22 &#181;m, polyethersulfone, 33 mm, gamma-sterilized</td>
			<td>Millipore Sigma</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrilotriacetic acid (NTA)</td>
			<td>Sigma</td>
			<td>72560-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 25 G x 5/8 in. hypodermic general use</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Strips, plastic pH5.0-9.0</td>
			<td>Fisher Scientific</td>
			<td>13-640-519</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades 0.22 mm</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (g)</td>
			<td>Mettler Toledo</td>
			<td>PB1502-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale (mg)</td>
			<td>Mettler Toledo</td>
			<td>Balance XS204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe, slip tip (1 mL)</td>
			<td>Fisher Scientific</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageLab</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SigmaPlot</td>
			<td>Systat</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitating iron transport across the mouse placenta in vivo using nonradioactive iron isotopes

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal iron endowment is entirely dependent on iron transfer across the placenta, and perturbations of this transfer can lead to adverse pregnancy outcomes. In mice, measurement of iron fluxes across the placenta traditionally relied on radioactive iron isotopes, a highly sensitive but burdensome approach. Stable iron isotopes (57Fe and 58Fe) offer a nonradioactive alternative for use in human pregnancy studies.
Under physiological conditions, transferrin-bound iron is the predominant form of iron taken up by the placenta. Thus, 58Fe-transferrin was prepared and injected intravenously in pregnant dams to directly assess placental iron transport and bypass maternal intestinal iron absorption as a confounding variable. Isotopic iron was quantitated in the placenta and mouse embryonic tissues by inductively coupled plasma mass spectrometry (ICP-MS). These methods can also be employed in other animal model systems of physiology or disease to quantify in vivo iron dynamics.

An earlier study using stable iron isotopes to measure iron transport demonstrated that maternal iron deficiency resulted in the downregulation of the placenta iron exporter, FPN4. FPN is the only known mammalian iron exporter, and the absence of FPN during development results in embryonic death before E9.529. To determine whether the observed decrease in FPN expression translated functionally to decreased placental iron transport, 58Fe-Tf was injected intravenou...

Watch this Scientific Journal Video about Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes at JoVE.com

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal ...

quantitating-iron-transport-across-mouse-placenta-vivo-using

Research

JoVE Journal

Biology

1.9K Views.  University of California, Los Angeles. This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described. 

Video: Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...