Efficient Reprogramming of Canine Peripheral Blood Mononuclear Cells into Induced Pluripotent Stem Cells.

Forced coexpression of the transcription factors Oct3/4, Klf4, Sox2, and c-Myc reprograms somatic cells into pluripotent stem cells (PSCs). Such induced PSCs (iPSCs) can generate any cell type of the adult body or indefinitely proliferate without losing their potential. Accordingly, iPSCs can serve as an unlimited cell source for the development of various disease models and regenerative therapies for animals and humans. Although canine peripheral blood mononuclear cells (PBMCs) can be easily obtained, they have a very low iPSC reprogramming efficiency. In this study, we determined the reprogramming efficiency of canine PBMCs under several conditions involving three types of media supplemented with small-molecule compounds. We found that canine iPSCs (ciPSCs) could be efficiently generated from PBMCs using N2B27 medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and a small-molecule cocktail (Y-27632, PD0325901, CHIR99021, A-83-01, Forskolin, and l-ascorbic acid). We generated five ciPSC lines that could be maintained in StemFit® medium supplemented with LIF. The SeVdp(KOSM)302L vectors were appropriately silenced in four ciPSC lines. Of the two lines characterized, both were positive for alkaline phosphatase activity and expressed pluripotency markers, including the Oct3/4, Sox2, and Nanog transcripts, as well as the octamer-binding transcription factor (OCT) 3/4 and NANOG proteins, and the SSEA-1 carbohydrate antigen. The ciPSCs could form embryoid bodies and differentiate into the three germ layers, as indicated by marker gene and protein expression. Furthermore, one ciPSC line formed teratomas comprising several tissues from every germ layer. Our ciPSC lines maintained a normal karyotype even after multiple passages. Moreover, our new reprogramming method was able to generate ciPSCs from multiple donor PBMCs. In conclusion, we developed an easy and efficient strategy for the generation of footprint-free ciPSCs from PBMCs. We believe that this strategy can be useful for disease modeling and regenerative medicine in the veterinary field.

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium.

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.

Generation of Hepatocytes from Pluripotent Stem Cells for Drug Screening and Developmental Modeling.

Hepatocytes produced from the differentiation of human pluripotent stem cells can be used to study human development and liver disease, to investigate the toxicological response of novel drug candidates, and as an alternative source of primary cells for transplantation therapies. Here, we describe a method to produce hepatocytes by differentiating human pluripotent stem cells into definitive endoderm, patterning definitive endoderm into anterior definitive endoderm, specifying anterior definitive endoderm into hepatic endoderm, and differentiating hepatic endoderm into immature hepatocytes. These cells are further matured in either two-dimensional or three-dimensional culture conditions to produce cells capable of metabolizing xenobiotics and generating liver-specific proteins, such as albumin and alpha 1 antitrypsin.

Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules.

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy, disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers, urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen, Chlorpromazine, Diclofenac, Digoxin, Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore, SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development.

Actl6a protects embryonic stem cells from differentiating into primitive endoderm.

Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern.

Human embryonic stem cell differentiation into insulin secreting ß-cells for diabetes.

hESC (human embryonic stem cells), when differentiated into pancreatic ß ILC (islet-like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five-step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine ß islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti-hyperglycaemic hormone-Glp1 (glucagon-like peptide 1) analogue Liraglutide with prolonged t(½) and Exendin 4. The differentiated islet-like 3D clusters expressed bonafide mature and functional ß-cell markers-PDX1 (pancreatic and duodenal homoeobox-1), C-peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C-peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D ß islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long-term in vivo functionally viable islets from hESC.

Expression of multiple stem cell markers in dental pulp cells cultured in serum-free media.

INTRODUCTION: Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS: Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS: Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS: SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.

Artificial human tissues from cord and cord blood stem cells for multi-organ regenerative medicine: viable alternatives to animal in vitro toxicology.

New medicinal products and procedures must meet very strict safety criteria before being applied for use in humans. The laboratory procedures involved require the use of large numbers of animals each year. Furthermore, such investigations do not always give an accurate translation to the human setting. Here, we propose a viable alternative to animal testing, which uses novel technology featuring human cord and cord blood stem cells. With over 130 million children born each year, cord and cord blood remains the most widely available alternative to the use of animals or cadaveric human tissues for in vitro toxicology.

Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells.

BACKGROUND & AIMS: New sources of beta cells are needed to develop cell therapies for patients with diabetes. An in vitro, sequential method has been developed to derive pancreatic progenitors, but this technique has not been used for other cell lines. We investigated whether definitive endoderm derived from human embryonic stem (hES) cells might be used to create beta cells. METHODS: Five hES cell lines were induced to form pancreatic progenitors and analyzed for pancreas markers. Cells were incubated with a bone morphogenetic protein (BMP) antagonist, retinoids, a Hedgehog antagonist, or fibroblast growth factor (FGF) and phenotypes were analyzed. RESULTS: Four hES cell lines sequentially generated definitive endoderm, primitive gut, and posterior foregut equivalents, as described previously. However, functional hepatocytes, rather than pancreas progenitors, developed. Consistent with liver development, FGF and BMP signaling pathways were involved in this process; their inhibition disrupted hepatocyte differentiation. During early stages of development, exposure of cells to noggin and retinoid acid, followed by FGF10, generated pancreatic cells (PDX1+; 50%-80%) that coexpressed FOXA2, HNF6, and SOX9. CONCLUSIONS: These findings demonstrate the combined functions of endogenous BMP and supplemented FGF in inducing differentiation of hepatocytes from hES cells and the ability to shift developmental pathways from hepatic to pancreatic cell differentiation. Although additional signals appear to be required for full specification of PDX1(+) early pancreatic progenitors (via PTF1a and NKX6.1 coexpression), these findings indicate the signaling pathways required for differentiation of bipotential progenitors.

CXCR4+/FLK-1+ biomarkers select a cardiopoietic lineage from embryonic stem cells.

Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi-lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue-specified progenitor cells. From a comprehensive pool of 11,272 quality-filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane-associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over-represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4(+) progenitors were sorted from the embryonic stem cell pool into mesoderm-restricted progeny according to co-expression with the early mesoderm marker Flk-1. In contrast to CXCR4(-)/Flk-1(-) cells, the CXCR4(+)/Flk-1(+) subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha-fetoprotein markers were all depleted. In fact, the CXCR4(+)/Flk-1(+) biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp-1, GATA-4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4(+)/Flk-1(+) transcriptome shared 97% identity with the CXCR4(-)/Flk-1(-) counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4(+)/Flk-1(+) progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage. Disclosure of potential conflicts of interest is found at the end of this article.

Conserved regulatory elements establish the dynamic expression of Rpx/HesxI in early vertebrate development.

TheRpx/Hesx1 homeobox gene is expressed during gastrulation in the anterior visceral and definitive endoderm and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland. This expression pattern suggests the presence of at least two distinct regulatory regions that control early and late Rpx transcription. Using transgenic mice, we have demonstrated that regulatory sequences in the 5' upstream region of Rpx are important for early expression in the anterior endoderm and neural plate and regulatory elements in the 3' region are required for late expression in Rathke's pouch. We have found that the genetically required LIM homeodomain-containing proteins Lim1/Lhx1 and Lhx3 are directly involved in the regulation of Rpx transcription. They bind two LIM protein-binding sites in the 5' upstream region of Rpx, which are required for Rpx promoter activity in both mice and Xenopus. Furthermore, we have found that a conserved enhancer in the 3' regulatory sequences of Rpx is not only required, but is also sufficient for the expression of Rpx transgenes in the developing Rathke's pouch.

Animal pole determinants define oral-aboral axis polarity and endodermal cell-fate in hydrozoan jellyfish Podocoryne carnea.

Cnidarians, in contrast with bilaterians, are generally considered to exhibit radial symmetry around a single body axis (oral-aboral) throughout their life-cycles. We have investigated how the oral-aboral axis is established in the hydrozoan jellyfish Podocoryne carnea. Vital labeling experiments showed that the oral end of the blastula derives from the animal pole region of the egg as has been demonstrated for other cnidarian species. Gastrulation is restricted to the oral pole such that the oral 20% of blastula cells give rise to endoderm. Unexpectedly, bisection experiments at the 8-cell stage showed that animal regions are able to develop into normally polarized larvae, but that vegetal (aboral) blastomeres completely fail to develop endoderm or to elongate. These vegetal-derived larvae also failed to polarize, as indicated by a lack of oral-specific RFamide-positive nerve cells and a disorganized tyrosinated tubulin-positive nerve net. A different result was obtained following bisection of the late blastula stage: aboral halves still lacked the capacity to develop endoderm but retained features of axial polarity including elongation of the larva and directional swimming. These results demonstrate for the first time in a cnidarian the presence of localized determinants responsible for axis determination and endoderm formation at the animal pole of the egg. They also show that axial polarity and endoderm formation are controlled by separable pathways after the blastula stage.

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice.

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.

Endo16, a large multidomain protein found on the surface and ECM of endodermal cells during sea urchin gastrulation, binds calcium.

Endo16 encodes a developmentally regulated protein restricted to cells participating in the formation of the archenteron during sea urchin gastrulation and to the stomach of the pluteus. The 4680-nt coding region of the Endo16 gene has been sequenced from overlapping cDNAs. Sequence analysis revealed that Endo16 is a large multidomain protein starting with a putative signal sequence at its amino terminus which is followed by a cysteine-rich region, two potential heparin-binding regions, an acidic domain of 5 clustered repeats, an RGD cell binding motif, and a group of 12 additional acidic repeats. Immunolocalization by confocal and electron microscopy demonstrate that the Endo16 protein is in the extracellular matrix and associated with the surface of endodermal cells in the mid and hindgut of the archenteron. The two distinct acidic repeat regions are similar to known calcium-binding sequences. A recombinant Endo16 protein containing both putative calcium-binding repeat regions has been shown to bind radioactive calcium. Tryptic digests of gastrula stage protein extracts in the presence and the absence of calcium have established that calcium stabilizes Endo16 protein against proteolytic degradation.

Sp1/egr-like zinc-finger protein required for endoderm specification and germ-layer formation in Drosophila.

Much of our present knowledge of the biological processes involved in pattern formation in Drosophila is derived from segmentation analysis. Comparatively little is known about the genetic requirement and mechanisms underlying the formation and separation of germ layers by morphogenetic movements during gastrulation. Here we show that the Drosophila gene huckebein (hkb), a member of the gap-gene class of segmentation genes, is required for germ-layer formation at blastoderm. Absence of the hkb product, an Sp1/egr-like zinc-finger protein, causes the ectodermal and mesodermal primordia to expand at the expense of endoderm anlagen. Conversely, ectopic expression of hkb inhibits the formation of the major gastrulation fold which gives rise to the mesoderm and prevents normal segmentation in the ectoderm. Thus, hkb is necessary for endoderm development and its activity defines spatial limits within the blastoderm embryo in which the germ layers are established.

Expression of cellular oncogenes in teratoma-derived cell lines.

The expression of ten proto-oncogenes was studied in cell lines derived from transplantable mouse teratomas. The cell lines represent different forms of early embryonic cell specialization. The analysis included two embryonal carcinoma (EC) lines (PCC3 and F9), and four differentiated cell lines derived from teratocarcinoma, namely trophoblastoma (3-TDM), parietal endoderm (PYS-2), visceral endoderm (PSA5-E) and skeletal myoblasts (Cl10). The expression of c-oncogenes was studied by analysing poly(A)+RNA for complementary sequences by dot blot and Northern blot hybridization. The results were related to the rate of cell multiplication and the state of differentiation by examining [3H]thymidine incorporation, growth curves and tissue-specific differentiation markers. Expression of c-myc and c-Ki-ras was found in all cell lines. In dot blot assays, poly(A)+RNA from all cell lines also hybridized with v-abl and v-sis probes. A marked decrease in c-myc expression was found in teratoma-derived myoblasts differentiating into myotubes. A similar reduction was found when 'nullipotent' F9 cells were induced by retinoic acid (RA) to form primitive endoderm. However, reduction of the growth rates of the parietal and visceral endodermal cell lines were not accompanied by decreased expression of c-myc or c-Ki-ras. Hybridization signals obtained with a v-sis probe was low in all teratoma-derived cell lines tested, except for the myogenic cell line Cl10. Both in exponentially growing and differentiated cultures of this line, two size classes of transcripts hybridized strongly to the v-sis probe. However, these transcripts, 7 and 3 kb, most likely represent endogenous retroviral transcripts and not c-sis transcripts. Expression of c-myb, c-mos, c-fes, c-src and c-erb A and c-erb B could not be detected in any of the cell lines studied.

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum.

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 microM retinoic acid are described. Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize alpha-fetoprotein but does secrete plasminogen activator. An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF. The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.

The annular hematoma of the shrew yolk-sac placenta.

The annular hematoma of the shrew, Blarina brevicauda, is a specialized portion of the yolk-sac wall. In this study, we have examined the fine structure of the different cellular components of the anular hematoma. Small pieces of the gestation sacs from seven pregnant shrews were fixed in glutaraldehyde and osmium tetroxide and processed for transmission electron microscopy. In the area of the trophoblastic curtain, the maternal capillary endothelial cells were hypertrophied and syncytial trophoblast surrounded the capillaries. Cellular trophoblast covered part of the luminal surface of the curtain region, whereas masses of apparently degenerating syncytium were present on other areas of the surface. Maternal erythrocytes, released into the uterine lumen from the curtain region, were phagocytized and degraded by the columnar cells of the trophoblastic annulus. No evidence of iron or pigment accumulation was evident in the parietal endodermal cells underlying the annular trophoblast. Parietal endodermal cells were characterized by cuboidal shape, widely dilated intercellular spaces, and cytoplasm containing granular endoplasmic reticulum. Endodermal cells of the visceral yolk-sac accumulated large numbers of electron-dense granules as well as glycogen in their cytoplasm. Hemopoietic areas and vitelline capillaries were found subjacent to the visceral endoderm. The various portions of the yolk-sac wall of Blarina appear to perform complementary functions which are probably important in maternal-fetal iron transfer.