Early Differentiation Markers Research Articles

Early life stress increases preadipocyte number and early markers of differentiation in female mice fed a high fat diet

Adipogenenic and lipogenenic functions are complex processes influenced by many mechanisms that become dysregulated with obesity. We have previously shown that Maternal Separation and Early Weaning (MSEW), a mouse model of early life stress, exacerbates high fat diet (HF)‐induced fat deposition in female mice, but not male mice. Therefore, the aim of this study was to determine the effects of MSEW on energy expenditure and on adipogenesis in response to diet‐induced obesity. MSEW and control mice were weaned either a low fat diet (LF, 10 Kcal from fat) or HF (60 % Kcal from fat) for 20 weeks. The adiposity of female MSEW mice fed a HF increased significantly compared to controls (42.09 ± 09 vs. 33.40±1.5 % body weight, respectively, P<0.05). The respiratory exchange ratio (RER), a function of macronutrient utilization and molecular interconversion, was not different between HF‐fed MSEW and control mice (0.84±0.03 vs. 0.83±0.01). In addition, MSEW and control females showed similar resting energy expenditure (REE, 0.50±0.02 vs. 0.47±0.02 kcal/hr), and food intake (38.6±1 vs. 41.03±1.0 kcals, respectively). Preadipocyte number was determined in subcutaneous white adipose tissue (scWAT) and gonadal WAT (gWAT) by cell sorting via flow cytometry. The percentage of CD45‐/CD31‐/CD34+/Sca1+ committed preadipocytes found in the stromal vascular fraction from scWAT was similar in MSEW and control mice (65.25±1.01 vs. 71.25±3.4 %, respectively). However, MSEW increased the total percentage of committed preadipocytes found in gWAT compared to controls (78.3±2.45 vs. 65.75±2.95%, respectively, p=0.063). In a subset of mice, fresh scWAT and gWAT were collagenase‐digested for adipocyte differentiation studies. Preadipocytes were harvested at day 0 of confluence (T0) or differentiated into mature adipocytes (after 8 days of differentiation, T8) to determine the expression of genes involved in early and late stage of adipocyte differentiation. At T0, the expression of leptin, a fat‐derived hormone from mature adipocytes, was significantly increased in scWAT from MSEW mice compared to controls (1.8 ± 0.3 vs. 0.65 ± 0.1 2^ddCT, p<0.05). At T8, female MSEW mice fed a HF showed increased CEBPa (1.13±0.4 vs 0.44 ±1.3 2^ddCT, p<0.05) and CEBPb (3.38±1.5 vs. 0.96±0.3 2^ddCT, p<0.05) and decreased PPARg mRNA (1.41±0.5 vs. 7.19±3.1 2^ddCT, p<0.05). Overall, MSEW‐mediated fat gain was not associated with decreased energy expenditure. However, chronologic expression of genes controlling the maturation process of preadipocytes from scWAT was altered, as well as the percentage of committed preadipocytes found in gWAT was increased from obese MSEW mice. Altogether, this data suggests that adipose tissue from MSEW mice may undergo a second cycle of differentiation more rapidly than control counterparts; this combined with the increased percentage of preadipocytes may be a possible origin of exacerbated HF‐induced obesity due to MSEW.Support or Funding InformationR01 HL135158 and R01 HL135158S1

Phenotype changes of subchondral plate osteoblasts based on a rat model of ovariectomy-induced osteoarthritis.

BackgroundOsteoarthritis (OA) is prevalent in postmenopausal women. Subchondral bone in ovariectomized (OVX) rats might play a more important role in cartilage degeneration compared with other types of OA. How subchondral osteoblast changes in OVX rats is still unclear. Understanding of osteoblast changes obtained from OVX subchondral bone might be helpful to clarify pathogenesis of OVX-OA.MethodsFemale Sprague-Dawley rats were randomly divided into two groups: Sham (n=20) and OVX (n=20). Serum levels of Alkaline phosphatase (ALP) and C-telopeptide of type I collagen (CTX-I) were measured every one or two weeks. All rats were executed at week 9 post surgery. The weight of rats and the wet weight of uterus were assessed. Micro-computed Tomography (micro-CT) was used to analyze the knee microstructure, and toluidine blue staining was employed to evaluate cartilage erosion. Subchondral osteoblast proliferation ability by cell counting kit-8 assay, osteogenic genes expressions by reverse transcription polymerase chain reaction (RT-PCR), differentiation and mineralization ability by ALP staining and alizarin red staining were evaluated and compared between Sham and OVX.ResultsOvariectomy induced significant increases of serum ALP and CTX-I as early as at week 2. At week 9 after surgery, the body weight of OVX rats was significantly increased, and uterus weight of OVX rats was remarkably decreased. OVX rats demonstrated significant subchondral bone change and cartilage erosion compared with Sham rats. mRNA levels of early markers of osteogenic differentiation (ALP, type I collagen, Runx2) were enhanced in OVX rats, but the late marker (osteocalcin) was not significantly different. ALP activity of osteoblasts increased, but the mineralization capacity decreased in OVX rats.ConclusionsSubchondral osteoblasts in OVX rats exhibited different proliferation, differentiation and mineralization abilities from normal counterparts.

Human skin-derived precursor cells xenografted in aganglionic bowel

PurposeOne in 5000 newborns is diagnosed with Hirschsprung disease each year in the United States. The potential of employing neural crest stem cells to restore the enteric nervous system has been investigated. Skin-derived precursor cells (SKPs) are multipotent progenitor cells that can differentiate into neurons and gliocytes in vitro and generate enteric ganglion-like structures in rodents. Here we examined the behavior of human SKPs (hSKPs) after their transplantation into a large animal model of colonic aganglionosis. MethodsJuvenile minipigs underwent a chemical denervation of the colon to establish an aganglionosis model. The hSKPs were generated from human foreskin and were cultured in neuroglial-selective medium. Cells were labeled with a fluorescent dye and were injected into the porcine aganglionic colon. After one week, transplanted hSKPs were assessed by immunofluorescence for markers of multipotency and neuroglial differentiation. ResultsIn culture, hSKPs expressed nestin and S100b indicative of neuroglial precursors. After xenografting in pigs, hSKPs were identified in the myenteric and submucosal plexuses of the colons. The hSKPs expressed nestin and early neuroglial differentiation markers. ConclusionsHuman SKPs transplanted into aganglionic colon demonstrated immunophenotypes of neuroglial progenitors, suggesting their potential use for Hirschsprung disease.Level of evidence

Bovine colostrum induces the differentiation of human primary keratinocytes.

Bovine colostrum, the first milk secreted by the mammary glands of cows shortly after they have given birth, provides a natural source of bioactive substances helpful to promote tissue development and repair, and to maintain a healthy immune system. Owing to its properties, the use of colostrum in the treatment of human diseases is under investigation. We evaluated the biological activity of colostrum on human primary keratinocytes, focusing on its effects with regard to a proliferation/differentiation balance. Using cellular and molecular approaches, we showed that colostrum favors a cell cycle withdrawal by increasing the expression of p21/WAF1 and p27/KIP1. It also promotes the transition of keratinocytes from a proliferating to a differentiating state, as assessed by a decrease in keratin 5 and an increase in keratin 16. We demonstrated the ability of colostrum to induce the expression of early and late differentiation markers (keratin 1, involucrin, and filaggrin) and the synthesis of caspase 14 and bleomycin hydrolase, the two main enzymes involved in filaggrin maturation. Moreover, we showed that bovine colostrum is able to promote keratinocyte stratification and terminal differentiation not only in two-dimensional (2D), but also in a more physiological system of three-dimensional (3D) skin equivalents. Finally, we demonstrated that colostrum stimulates cell differentiation through the PI3K/PLC-γ1/PKCα pathways mainly associated to tyrosine kinase receptors. These results suggest the possibility to benefit from colostrum properties for the treatment of skin diseases characterized by altered differentiation and perturbed barrier function.

A Novel DNA Methyltransferase Dnmt3a3 Splice Variant Represses Preadipocyte Proliferation and Differentiation.

Proliferation and differentiation of preadipocyte are essential for the formation of fat tissues. However, the genes that regulate the early stage of preadipocyte differentiation in chicken have remained elusive. Here we identify a novel spliced variant of the DNA methyltransferase Dnmt3a gene, named Dnmt3a3, that controls early preadipocyte differentiation. Dnmt3a3 expression is increased at the onset of preadipocyte differentiation and remains elevated during differentiation. Overexpression of Dnmt3a3 in preadipocytes markedly inhibits proliferation and cell-cycle progression, and this is accompanied by inhibition of the mRNA and protein level of cell-cycle control genes, such as p21 and p27. In addition, forced expression of Dnmt3a3 in differentiating preadipocytes represses early preadipocyte differentiation, and this was found to be accompanied by inhibition of the mRNA expression levels of early preadipocyte differentiation markers, such as GATA2, GATA3, C/EBPα, C/EBPβ, AP2, and PPARγ, or the protein levels of GATA3, C/EBPβ, and PPARγ. Taken together, these data demonstrate the participation of Dnmt3a3 in the proliferation and differentiation process of chicken primary preadipocyte cells.

Oligomers of the lipodystrophy protein seipin may co-ordinate GPAT3 and AGPAT2 enzymes to facilitate adipocyte differentiation

Seipin deficiency causes severe congenital generalized lipodystrophy (CGL) and metabolic disease. However, how seipin regulates adipocyte development and function remains incompletely understood. We previously showed that seipin acts as a scaffold protein for AGPAT2, whose disruption also causes CGL. More recently, seipin has been reported to promote adipogenesis by directly inhibiting GPAT3, leading to the suggestion that GPAT inhibitors could offer novel treatments for CGL. Here we investigated the interactions between seipin, GPAT3 and AGPAT2. We reveal that seipin and GPAT3 associate via direct interaction and that seipin can simultaneously bind GPAT3 and AGPAT2. Inhibiting the expression of seipin, AGPAT2 or GPAT3 led to impaired induction of early markers of adipocyte differentiation in cultured cells. However, consistent with normal adipose mass in GPAT3-null mice, GPAT3 inhibition did not prevent the formation of mature adipocytes. Nonetheless, loss of GPAT3 in seipin-deficient preadipocytes exacerbated the failure of adipogenesis in these cells. Thus, our data indicate that GPAT3 plays a modest positive role in adipogenesis and argue against the potential of GPAT inhibitors to rescue white adipose tissue mass in CGL2. Overall, our study reveals novel mechanistic insights regarding the molecular pathogenesis of severe lipodystrophy caused by mutations in either seipin or AGPAT2.

MicroRNA-184 is induced by store-operated calcium entry and regulates early keratinocyte differentiation.

Extracellular calcium (Ca2+ ) and store-operated Ca2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

Inhibition of sphingosine 1-phosphate lyase activates human keratinocyte differentiation and attenuates psoriasis in mice

Sphingosine 1-phosphate (S1P) lyase is an intracellular enzyme that catalyzes the irreversible degradation of S1P and has been suggested as a therapeutic target for the treatment of psoriasis vulgaris. Because S1P induces differentiation of keratinocytes, we examined whether modulation of S1P lyase and altered intracellular S1P levels regulate proliferation and differentiation of human neonatal epidermal keratinocyte (HEKn) cells. To identify the physiological functions of S1P lyase in skin, we inhibited S1P lyase in HEKn cells with an S1P lyase-specific inhibitor (SLI) and with S1P lyase 1 (SGPL1)-specific siRNA (siSGPL1). In HEKn cells, pharmacological treatment with the SLI caused G1 arrest by upregulation of p21 and p27 and induced keratin 1, an early differentiation marker. Similarly, genetic suppression by siSGPL1 arrested the cell cycle at the G1 phase and activated differentiation. In addition, enzyme suppression by siSGPL1 upregulated keratin 1 and differentiation markers including involucrin and loricrin. When hyperproliferation of HEKn cells was induced by interleukin (IL)-17 and IL-22, pharmacologic inhibition of S1P lyase by SLI decreased proliferation and activated differentiation of HEKn cells simultaneously. In addition, SLI administration ameliorated imiquimod-induced psoriatic symptoms including erythema, scaling, and epidermal thickness in vivo. We thus demonstrated that S1P lyase inhibition reduces cell proliferation and induces keratinocyte differentiation, and that inhibition may attenuate psoriasiform changes. Collectively, these findings suggest that S1P lyase is a modulating factor for proliferation and differentiation, and support its potential as a therapeutic target for psoriasis in human keratinocytes.

Insight into Nephrocan Function in Mouse Endoderm Patterning.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

A retrospective case control study identifies peripheral blood mononuclear cell albumin RNA expression as a biomarker for non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) improves after bariatric surgery. The aim of this study was to determine whether peripheral blood mononuclear cell albumin gene expression was related to NAFLD and whether albumin (ALB) and alpha fetoprotein (AFP) expression could be detected in whole blood and visceral adipose tissue. Using a retrospective case control study design, RNA isolated from peripheral blood mononuclear cells from patients prior to undergoing bariatric surgery was used for pooled microarray analysis. Quantitative polymerase chain reaction (QPCR) was used to analyze whole blood and visceral adipose tissue. Liver histology was obtained via intra-operative biopsy and clinical data extracted from the electronic health record. The albumin (ALB) gene was the second most up-regulated found in microarray analysis of peripheral blood mononuclear cell RNA from patients with hepatic lobular inflammation versus normal liver histology. Transcript levels of ALB were significantly different across those with normal (n = 50), steatosis (n = 50), lobular inflammation (n = 50), and peri-sinusoidal fibrosis (n = 50) liver histologies, with lobular inflammation 3.9 times higher than those with normal histology (p < 0.017). Albumin expression levels decreased in 11/13 patients in paired samples obtained prior to and at 1year after Roux-en-Y gastric bypass surgery. ALB expression could be detected in 23 visceral adipose tissue samples obtained intra-operatively and in 18/19 available paired whole blood samples. No significant correlation was found between ALB expression in visceral adipose tissue and whole blood RNA samples. Alpha fetoprotein expression as a marker of early hepatocytic differentiation was detected in 17/17 available VAT RNA samples, but in only 2/17 whole blood RNA samples. Albumin RNA expression from blood cells may serve as a biomarker of NAFLD. Albumin and alpha fetoprotein appear to be ubiquitously expressed in visceral adipose tissue in patients with extreme obesity.

7-O-methylpunctatin, a Novel Homoisoflavonoid, Inhibits Phenotypic Switch of Human Arteriolar Smooth Muscle Cells.

Remodeling of arterioles is a pivotal event in the manifestation of many inflammation-based cardio-vasculopathologies, such as hypertension. During these remodeling events, vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. The latter is characterized by increased proliferation, migration, and invasion. Compounds with anti-inflammatory actions have been successful in attenuating this phenotypic switch. While the vast majority of studies investigating phenotypic modulation were undertaken in VSMCs isolated from large vessels, little is known about the effect of such compounds on phenotypic switch in VSMCs of microvessels (microVSMCs). We have recently characterized a novel homoisoflavonoid that we called 7-O-methylpunctatin (MP). In this study, we show that MP decreased FBS-induced cell proliferation, migration, invasion, and adhesion. MP also attenuated adhesion of THP-1 monocytes to microVSMCs, abolished FBS-induced expression of MMP-2, MMP-9, and NF-κB, as well as reduced activation of ERK1/2 and FAK. Furthermore, MP-treated VSMCs showed an increase in early (myocardin, SM-22α, SM-α) and mid-term (calponin and caldesmon) differentiation markers and a decrease in osteopontin, a protein highly expressed in synthetic VSMCs. MP also reduced transcription of cyclin D1, CDK4 but increased protein levels of p21 and p27. Taken together, these results corroborate an anti-inflammatory action of MP on human microVSMCs. Therefore, by inhibiting the synthetic phenotype of microVSMCs, MP may be a promising modulator for inflammation-induced arteriolar pathophysiology.

Retinal differentiation in an altricial bird species, Taeniopygia guttata: An immunohistochemical study

The bird retina offers an excellent model to investigate the mechanisms that coordinate the morphogenesis, histogenesis, and differentiation of neuron and glial cells. Although these developmental features have been intensively studied in the chicken (Gallus gallus, Linnaeus 1758), a precocial bird species, little is known about retinogenesis in altricial birds. The purpose of this study was to examine the differentiation of retinal cells in the altricial zebra finch (Taeniopygia guttata, Vieillot, 1817) and compare the results with those from previous studies in G. gallus. By using immunohistochemical techniques, the first differentiated TUJ1-/Isl1-positive neuroblasts were detected in the vitreal surface of the neuroblastic layer at later incubation times in T. guttata than in G. gallus (108 h vs 55 h). The immunoreactivity of these early differentiation markers coincided temporo-spatially with the appearance of the first PCNA-negative nuclei. Furthermore, the first visinin-positive photoreceptors (132 h vs 120 h) and the first Prox-1-immunoreactive neuroblasts (embryonic day 7.25 (E7.25) vs E6.5) were also detected at later embryonic stages in the retina of T. guttata than in the retina of G. gallus. At E13, one day before hatching, abundant PCNA- and pHisH3-immunoreactivities were detected in the T. guttata retina, while proliferation was almost absent in the G. gallus retina at perinatal stages. Therefore, these results suggest that cell differentiation in the retina is delayed in the altricial bird compared to precocial birds. Furthermore, the T. guttata retina was not completely developed at hatching, and abundant mitotically active precursor cells of retinal neurons were found, suggesting that retinal neurogenesis was intense at perinatal stages.

Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model

BackgroundHirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. MethodsJuvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. ResultsPrior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. ConclusionsAutologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. Type of StudyBasic science.

Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin.

Graphene foam holds promise for tissue engineering applications. In this study, graphene foam was used as a three-dimension scaffold to evaluate cell attachment, cell morphology, and molecular markers of early differentiation. The aim of this study was to determine if cell attachment and elaboration of an extracellular matrix would be modulated by functionalization of graphene foam with fibronectin, an extracellular matrix protein that cells adhere well to, prior to the establishment of three-dimensional cell culture. The molecular dynamic simulation demonstrated that the fibronectin–graphene interaction was stabilized predominantly through interaction between the graphene and arginine side chains of the protein. Quasi-static and dynamic mechanical testing indicated that fibronectin functionalization of graphene altered the mechanical properties of graphene foam. The elastic strength of the scaffold increased due to fibronectin, but the viscoelastic mechanical behavior remained unchanged. An additive effect was observed in the mechanical stiffness when the graphene foam was both coated with fibronectin and cultured with cells for 28 days. Cytoskeletal organization assessed by fluorescence microscopy demonstrated a fibronectin-dependent reorganization of the actin cytoskeleton and an increase in actin stress fibers. Gene expression assessed by quantitative real-time polymerase chain reaction of 9 genes encoding cell attachment proteins (Cd44, Ctnna1, Ctnnb1, Itga3, Itga5, Itgav, Itgb1, Ncam1, Sgce), 16 genes encoding extracellular matrix proteins (Col1a1, Col2a1, Col3a1, Col5a1, Col6a1, Ecm1, Emilin1, Fn1, Hapln1, Lamb3, Postn, Sparc, Spp1, Thbs1, Thbs2, Tnc), and 9 genes encoding modulators of remodeling (Adamts1, Adamts2, Ctgf, Mmp14, Mmp2, Tgfbi, Timp1, Timp2, Timp3) indicated that graphene foam provided a microenvironment conducive to expression of genes that are important in early chondrogenesis. Functionalization of graphene foam with fibronectin modified the cellular response to graphene foam, demonstrated by decreases in relative gene expression levels. These findings illustrate the combinatorial factors of microscale materials properties and nanoscale molecular features to consider in the design of three-dimensional graphene scaffolds for tissue engineering applications.

Cerebral Palsy: Early Markers of Clinical Phenotype and Functional Outcome.

The Prechtl General Movement Assessment (GMA) has become a cornerstone assessment in early identification of cerebral palsy (CP), particularly during the fidgety movement period at 3–5 months of age. Additionally, assessment of motor repertoire, such as antigravity movements and postural patterns, which form the Motor Optimality Score (MOS), may provide insight into an infant’s later motor function. This study aimed to identify early specific markers for ambulation, gross motor function (using the Gross Motor Function Classification System, GMFCS), topography (unilateral, bilateral), and type (spastic, dyskinetic, ataxic, and hypotonic) of CP in a large worldwide cohort of 468 infants. We found that 95% of children with CP did not have fidgety movements, with 100% having non-optimal MOS. GMFCS level was strongly correlated to MOS. An MOS > 14 was most likely associated with GMFCS outcomes I or II, whereas GMFCS outcomes IV or V were hardly ever associated with an MOS > 8. A number of different movement patterns were associated with more severe functional impairment (GMFCS III–V), including atypical arching and persistent cramped-synchronized movements. Asymmetrical segmental movements were strongly associated with unilateral CP. Circular arm movements were associated with dyskinetic CP. This study demonstrated that use of the MOS contributes to understanding later CP prognosis, including early markers for type and severity.

602 Sox13 is a novel marker for hair follicle development and differentiation

The Sry-related high-mobility-group box (Sox) gene encodes a transcription factor family that plays crucial roles in the determination of cell fate and organogenesis. Sox13, a group D member of the Sox family, is expressed in various tissues such as brain, cartilage, hair, kidney, pancreas, liver, kidney and blood vessels. However, knowledge of the biological roles of Sox13 is largely limited. To examine the precise expression domains of Sox13 protein and its role in mouse development, we generated Sox13-LacZ-knock-in mice (Sox13LacZ/+), in which the Sox13 coding sequence was disrupted by an in-frame insertion of the b-galactosidase (LacZ) gene. Using the knock-in mice, we examined spatiotemporal expression patterns of Sox13 in the hair follicle and its role in hair development. X-gal staining in Sox13LacZ/+ embryos revealed that Sox13 is initially expressed in the epithelial portion of the placode at E14.5, and subsequently in the hair germ and the hair peg during hair follicle morphogenesis. Immunohistochemistry with an anti-b-galactosidase antibody and anti-hair keratin antibodies that specifically mark the different layers of the hair follicle revealed that during the postnatal phases of hair development Sox13 was predominantly expressed in the outer root sheath. However, the integumentary structures of Sox13LacZ/LacZ mice were grossly and histologically normal. These results suggest that although Sox13 is dispensable for epidermal and adnexal development, Sox13 is a novel marker for early hair follicle development and differentiation.

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Substrate softness promotes terminal differentiation of human keratinocytes without altering their ability to proliferate back into a rigid environment.

Substrate stiffness is a key regulator of cell behavior. To investigate how mechanical properties of cell microenvironment affect the human keratinocyte, primary cells were seeded on polyacrylamide hydrogels of different compliances (soft: 4kPa, medium: 14kPa, rigid: 45kPa) in comparison with glass coverslip (> GPa). Keratinocyte spreading and proliferation were strongly decreased on the softest hydrogel, while no significant difference was observed between medium, rigid hydrogels and glass coverslip, and cells' viability was comparable in all conditions after 72h. We then performed a RNA-seq to compare the transcriptomes from keratinocytes cultured for 72h on the softest hydrogel or on coverslips. The cells on the soft hydrogel showed a strong increase in the expression of late differentiation marker genes from the epidermal differentiation complex (1q21) and the antioxidant machinery. In parallel, these cells displayed a significant loss of expression of the matrix receptors (integrin α6 and β1) and the EGF receptor. However, when these cells were replated on a plastic culture plate (> GPa), they were able to re-engage the proliferation machinery with a strong colony-formation efficiency. Overall, using low-calcium differentiation monolayers at confluence, the lesser the rigidity, the stronger the markers of late differentiation are expressed, while the inverse is observed regarding the markers of early differentiation. In conclusion, below a certain rigidity, human keratinocytes undergo genome reprogramming indicating terminal differentiation that can switch back to proliferation in contact with a stiffer environment.

Bone Morphogenetic Protein 2-Conjugated Silica Particles Enhanced Early Osteogenic Differentiation of Adipose Stem Cells on the Polycaprolactone Scaffold.

Silica particles (SPs) induce cell proliferation and osteogenic differentiation. We reported that SPs in the scaffold induced early stage osteogenic differentiation. A polycaprolactone (PCL) scaffold was fabricated with a 10wt% SPs. The surface of PCL scaffold was coated with a 10µg/mL collagen solution. Next, the scaffold was conjugated with 2μM SPs, 2μg/mL bone morphogenetic protein 2 (BMP2), or 2μM BMP2-conjugated SPs (BCSPs). Green fluorescent protein-coupled BMP2 was applied to fabricate the scaffold. The fluorescence intensity was analyzed by confocal microscopy. The mRNA levels of the early osteogenic differentiation marker, alkaline phosphatase (ALP), were analyzed by real-time quantitative polymerase chain reaction. Levels of BMP2, RUNX2, ERK1/2, and AKT were assessed by western blotting. ALP mRNA levels were significantly higher in the BCSP-conjugated scaffold than in the other scaffolds. In the early stage of osteogenic differentiation, the protein levels of BMP2, RUNX2, ERK1/2, and AKT in cells were significantly higher in the BCSP-conjugated scaffold than in other scaffolds. Thus, the BCSP composite scaffold induced rapid osteogenic differentiation. These results suggest that BCSP composite can be used to promote early stage osteogenic differentiation and show promise as a material for use in scaffolds for bone regeneration.

Characterization and in vitro evaluation of gelatin–chitosan scaffold reinforced with bioceramic nanoparticles for bone tissue engineering

Abstract