Characterization and differentiation of human first trimester placenta trophoblastic cells in culture.

A preparation of highly enriched isolated cytotrophoblasts was obtained from first trimester placenta using dispase incubation of villous tissue at 4 degrees C, followed by a spontaneous cell release at 37 degrees C. After 24 h of culture, 90-95% of the cells were immunostained by anticytokeratin antibody, showing their epithelial characteristic. After 48 h of culture, these cells differentiated into syncytiotrophoblast, as shown by optic and electron microscopic study. The secretion of hCG, and of its free alpha and beta subunits, and the secretion of hPL were studied as a function of cell culture time. While the level of secreted hCG and its free subunits was stable during 72 h of culture, the hPL level was undetectable during the first 48 h of culture, increasing continuously afterwards. Addition of dibutyryl cAMP from the start or after 96 h of cell culture induced an increase of hCG production and of its free subunits and also stimulated the secretion of hPL. This suggests that these cells maintained the capacity to respond to stimuli which increased intracellular cAMP level. Such a cell culture is of interest in further determining the mechanisms of early gestation involved in the differentiation and growth of placental cytotrophoblasts, and in the regulation of their endocrine functions.     

Transcriptional products of the human placental lactogen gene

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.     

Catalytic and regulatory roles of species involved in metal–nucleotide equilibriums in human pyridoxal kinase

Pyridoxal 5′-phosphate is the active form of vitamin B₆ and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal–nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn²⁺ the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP^4? and HATP^3? did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP^2?, although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn²⁺; as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC₅₀ of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal–nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.     

人胎盘催乳素多型性研究

通过纯化和分析人胎盘催乳素,发现其有两个等电点,与文献报道不一致,利用等电聚焦制备电泳分离了两个等电点组分。免疫学活性测定的结果表明,两个组分均有活性,且活性有差别,等电点高的组分,免疫学活性强。通过Ｎ端氨基酸分析发现,两组分的Ｎ端均为缬氨酸。推测人胎盘催乳素存在亚型,为进一步研究人胎盘催乳素的结构和功能以及临床上正确地利用此激素提供了有价值的依据。     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

Synthesis of human placental lactogen messenger RNA as a function of gestation.

It was shown previously that 4 to 5 times more human placental lactogen (hPL) was synthesized in cell-free extracts from term placentae than in comparable extracts prepared from first trimester tissue. In an attempt to define what accounts for this differential rate of synthesis RNA was prepared from first trimester and term placentae. Following purification through an oligo(dT)-cellulose column, these RNA preparations were tested for their ability to direct the synthesis of the hPL precursor in the wheat germ cell-free system. With similar amounts of first trimester and term mRNA, the overall efficiency as defined by the stimulation of total amino acid incorporation was comparable. However, there was approximately 4 times more hPL synthesized in the presence of term RNA. This was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic fingerprinting. The peak of the hPL precursor messenger activity sedimented at 12 to 13 S on sucrose gradient. Analysis of the RNA by formamide-polyacrylamide gel electrophoresis further supported this value. The data indicate that the increased synthesis of hPL at term reflects greater levels of hPL mRNA in term tissue than in first trimester tissue. The data also show that the overall in vivo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in the greater proportion of hPL synthesized per g of tissue. The latter results from the continual differentiation of the placenta occurring throughout gestation.     

Crystal structure and site 1 binding energetics of human placental lactogen

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.     

Arachidonic acid stimulates 45calcium efflux and hPL release in isolated trophoblast cells

Previous investigations from this laboratory have indicated that arachidonic acid stimulates a rapid, dose-dependent and reversible increase in hPL release which is not dependent on cyclooxygenase or lipoxygenase metabolism. To investigate further the mechanism by which arachidonic acid stimulates the release of hPL, the effect of arachidonic acid on the release of 45Ca from perifused cells prelabelled with 45CaCl was examined in an enriched cell culture population of term human syncytiotrophoblast. Arachidonic acid (10-100 microM) stimulated a dose-dependent, rapid, and reversible increase in the release of both 45Ca and hPL from the perifused placental cells. On the other hand, palmitic acid had little effect on either hPL release or 45Ca release even at concentrations as high as 100 microM. Ionophore A23187 (1-10 microM) also stimulated a dose-dependent and reversible increase in hPL release. Since arachidonic acid increases the mobilization of cellular calcium, as reflected by the increased 45calcium efflux, and since an increase in the intracellular calcium concentration appears to stimulate an increase in hPL release, these results suggest that the stimulation of hPL release by arachidonic acid may be due, at lease in part, to the effects of the fatty acid on cellular calcium mobilization.     

Preservation of human chorionic gonadotropin and placental lactogen secretion and normal morphology following long-term culture of normal human placental cells

In order to study the regulation of hCG and hPL secretion during gestation, a system for the preservation of the functional integrity of normal placental cells in long-term culture was established. Normal term placental cells were dispersed with 0.25% trypsin-500 units DNAse I and cultured in a monolayer in Dulbecco's modified Eagle medium with 10% fetal bovine serum. Normal cell morphology, basal hCG and hPL production and hCG responses to dibutyryl cAMP were preserved till 54 days of culture. This model may be useful for the study of long-term regulation of normal placental hCG and hPL synthesis and secretion.     

Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.     

Gaps in the knowledge of human platelet lysate as a cell culture supplement for cell therapy: a joint publication from the AABB and the International Society for Cell & Gene Therapy

Fetal bovine serum (FBS) is used as a growth supplement in a wide range of cell culture applications for cell-based research and therapy. However, as a xenogenic product, FBS can potentially transmit prions and adventitious viruses as well as induce undesirable immunologic reactions. In addition, the use of bovine fetuses for FBS production raises concerns as society looks for ways to replace animal testing and reduce the use of animal products for scientific purposes, in particular for the manufacture of clinical products intended for human use. Until chemically defined media are available for these purposes, human platelet lysate (hPL) has been introduced as an attractive alternative for replacing FBS as a cell culture supplement. hPL is a human product that can be produced from outdated platelets avoiding ethical, medical and animal welfare concerns. An increasing number of studies demonstrate that hPL can promote cell growth similarly or even better than FBS in specific cell types. Due to increasing interest in hPL, the AABB and the International Society of Cell Therapy (ISCT) established a joint working group to address its potential. With this article, we aim to present an overview of hPL, identifying the gaps in information on how hPL is produced and tested and the barriers to its translational use in the production of clinical-grade cell therapy products.     

Placental Lactogen Is Expressed but Is Not Translated into Protein in Breast Cancer

Introduction

Several studies reported that the pregnancy-specific hormone placental lactogen (hPL) is expressed at both mRNA and protein levels in breast cancer. The overall objective was to establish hPL, the product of the CSH1 and CSH2 genes, as a biomarker for breast cancer.

Methods

CSH expression was determined at the mRNA level in breast cancer cell lines (BCC) and primary carcinomas by real-time and conventional PCR and the products verified as CSH1 by sequencing. Expression of hPL protein was examined by western blots and immuno-histochemistry, using commercial and custom-made polyclonal and monoclonal antibodies.

Results

Variable levels of CSH mRNA were detected in several BCC, and in some primary tumors. We detected a protein, slightly larger than recombinant hPL by western blotting using several antibodies, leading us to postulate that it represents an hPL variant (‘hPL’). Furthermore, some monoclonal antibodies detected ‘hPL’ by immunohistochemistry in breast carcinomas but not in normal breast. However, further examination revealed that these antibodies were non-specific, as efficient suppression of CSH mRNA by shRNA did not abolish the ‘hPL’ band. Custom-made monoclonal antibodies against recombinant hPL detected hPL of the correct size in placental lysate and hPL-overexpressing BCC, but not in unmodified cells or primary carcinomas. hPL protein was detected only when mRNA was increased several thousand fold.

Conclusions

We call into question previous reports of hPL expression in breast cancer which relied on mRNA levels as surrogates for protein and/or used improperly validated antibodies to measure hPL protein levels. Our data suggests that an inhibitory mechanism(s) prevents translation of CSH mRNA in breast cancer when not highly expressed. The mechanism by which translation of CSH mRNA is inhibited is intriguing and should be further investigated.     

Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+). Other monovalent cations, Li(+), Cs(+), and Rb(+) do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 A resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg(2+) and Na(+) acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.

In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.     

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations

Background aims

Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported.

High molecular weight forms of human placental lactogen: synthesis in vitro and binding to membrane receptors for lactogenic hormones.

Translation in wheat germ extracts of poly(A)-containing RNA isolated from human term placentas resulted in the synthesis of immunoreactive forms of human placental lactogen (hPL) capable of specific binding to lactogenic receptors. The minor component coelectrophoresed on sodium dodecyl sulfate-polyacrylamide gels with authentic hPL while the major component migrated with an apparent molecular weight about 3000 larger. In addition to this precursor-like molecule, even higher molecular weight forms of hPL were observed under certain conditions: (i) when the cell-free translation products were purified by precipitation with anti-hPL serum followed by dissociation of the immunoprecipitate in guanidine hydrochloride and chromatography of the solubilized material on Sephadex G-150 in the same denaturing buffer, and (ii) when the cell-free reaction mixture was analyzed by direct chromatography on Sephadex G-150 in nondenaturing buffers. Under both sets of conditions 50–75% of the radioactivity was eluted in the column void volume, suggesting it had a molecular weight of 150,000 or more. When the high molecular weight translated product was analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the radioactive components were identical to authentic hPL and the precursorlike form, suggesting the large forms are aggregates of the smaller forms. Both the very high molecular weight forms, composed primarily of the precursor-like molecule, and the less aggregated products bound to specific lactogenic hormone receptors in rat liver membrane preparations, although the larger forms exchanged less readily with unlabeled hPL than did the monomeric form of the hormone. The aggregated, receptor-bindable cell-free translation product may be similar to high molecular weight lactogens previously described in vivo.