The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells.

Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.

Adipose-derived stem cells and BMP2: part 1. BMP2-treated adipose-derived stem cells do not improve repair of segmental femoral defects.

Recombinant human bone morphogenetic protein-2 (rhBMP2) has been shown to induce both in vitro osteogenic differentiation and in vivo bone formation, with the capacity of rhBMP2 to elicit the repair of numerous bony defects (calvaria, spinal fusion, femora, and so on) well documented. In addition, rhBMP2 has been approved by the Food and Drug Administration (FDA) for selected human indications. Despite the fact that healing is often achieved, the challenge still remains to optimize the therapeutic use of rhBMP2. One avenue may be through the combination of rhBMP2 with stem cells capable of osteogenic differentiation. This study investigates the ability of rhBMP2 at various doses in combination with human adipose-derived stem cells (ASCs) to heal critical-sized rat segmental femoral defects. For this, different doses of rhBMP2 were incorporated with apatite-coated porous poly(l-lactide-co-dl-lactide) (70 : 30) (PLDLA) scaffolds, seeded with ASCs, and implanted into athymic rats. After 8 weeks, all implants were harvested and processed for bone formation using micro computed tomography (microCT) analysis and histology. Despite the findings that indicate no adverse effect of the apatite surface on ASC osteogenesis, no significant difference in bone formation could be qualitatively or quantitatively determined upon the implantation of ASC-seeded scaffolds absorbed to increasing doses of rhBMP2. Such results would suggest that the presence of ASCs within rhBMP2-absorbed scaffolds does not improve the bone-forming ability of the construct and that the formation of bone may be driven by the rhBMP2 alone. Based on these results, the addition of ASCs to rhBMP2-treated scaffolds may provide no significant advantage in terms of the ability to heal bone.

Adipose-derived stem cells and BMP2: part 2. BMP2 may not influence the osteogenic fate of human adipose-derived stem cells.

Although recent studies have proposed that human adipose-derived stem cells (ASCs), together with BMP2, can heal critical-sized bony defects, a companion study in this issue suggests that ASCs may not respond to BMP2 in vivo. To examine why this may be occurring. ASCs were treated with BMP2 and the cells' in vitro osteogenic capacity assessed along with the canonical BMP2 signaling pathway. In vitro treatment of ASCs with BMP2 had no consistent and significant effect on matrix mineralization or their expression of several osteogenic markers. Consistent and significant changes to Smad1/5/8 phosphorylation levels were also not observed upon BMP2 induction. The removal of dexamethasone from the BMP2 induction conditions had no effect on the observed results nor did stimulating ASCs with BMP2 from an alternate source (INFUSE; Medtronic, Minneapolis, MN, USA). In addition, no BMP-induced nuclear translocation of Smad1/Smad4 complexes could be discerned, suggesting that the canonical BMP2 signaling pathway may not be functional in ASCs. Interestingly, three downstream BMP2 pathway genes, distal-less3 (dlx3), distal-less5 (dlx5), and osterix, were not expressed in BMP2-induced ASCs, calling the utility of BMP2 induced in ASCs into question. The results of this in vitro study were consistent with that of our companion in vivo study that suggests a lack of effect of BMP2 on the osteogenic capacity of ASCs. Taken together, the data from both studies suggest that ASC osteogenic differentiation may not be influenced by BMP2. Consequently, combining BMP2 treatment with adult stem cells, like ASCs, may not be a viable strategy for bony healing.

MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects.

Numerous growth factors, peptides, and small molecules are being developed for bone tissue engineering. The optimal dosing, stability, and bioactivity of these biological molecules are likely influenced by the carrier biomaterial. Efficient evaluation of various formulations will require objective evaluation of in vitro culture systems and in vivo regeneration models. The objective of this paper is to examine the utility of microcomputed tomography (microCT) over conventional techniques in the evaluation of the bone morphogenetic protein-2 (BMP-2) dose response effect in a three-dimensional (3D) in vitro culture system and in an established calvarial defect model. Cultured MC3T3-E1 osteoblasts displayed increased cellular density, extracellular matrix (ECM) production, and mineralization on 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds in a BMP-2 dose dependent manner. MicroCT revealed differences in shape and spatial organization of mineralized areas, which would not have been possible through conventional alizarin red staining alone. Additionally, BMP-2 (doses of 30 to 240 ng/mm(3)) was grafted into 5 mm critical sized rat calvarial defects, where increased bone regeneration was observed in a dose dependent manner, with higher doses of BMP-2 inducing greater bone area, volume, and density. The data revealed the utility of microCT analysis as a beneficial addition to existing techniques for objective evaluation of bone tissue engineering and regeneration.

Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression.

The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5 x SBF) at 37 degrees C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.

Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover.

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

In vitro response of MC3T3-E1 pre-osteoblasts within three-dimensional apatite-coated PLGA scaffolds.

Biomimetic apatites have been reported to promote osteogenic activities in numerous in vivo and in vitro models, but the precise mechanism by which the apatite microenvironment promotes such activities is not well understood. Such mechanistic studies require reproducible model systems that are relevant to tissue engineering practices. Although two-dimensional (2D) apatite-coated polystyrene culture dishes provide practicality and reproducibility, they do not simulate the effects of the three-dimensional (3D) microenvironment and degrading polymeric substrates. A simple 3D model system to address these relevant effects, and its utilization in the investigation of apatite-promoted osteoblastic differentiation in vitro is reported in this paper. Apatite coating was achieved by sequentially immersing poly(lactide-co-glycolide) (PLGA) scaffolds into different simulated body fluids (SBF). SEM, EDX, FTIR, TEM electron diffraction confirmed the apatite coating to comprise of calcium-deficient carbonated hydroxyapatite crystals. While both apatite-coated and non-coated PLGA scaffolds supported MC3T3-E1 attachment, spreading, and proliferation, significant differences in osteoblastic differentiation were observed. Relative to non-coated controls, quantitative real-time PCR revealed significant apatite-associated suppression of alkaline phosphatase (ALP), early upregulation of osteopontin (OPN) at 3 days, and upregulation of osteocalcin (OCN) and bone sialoprotein (BSP) at 4 weeks. In summary, apatite-promoted osteoblastic differentiation can be observed in a 3D model system that is relevant to tissue engineering.

Gelatin-embedded cell-polymer constructs for histological cryosectioning.

Many tissue-engineering strategies involve the delivery of cells via porous polymer scaffolds. Obtaining histological sections of the emerging tissue is often necessary to analyze numerous characteristics of the microscopic environment. However, difficulties arise upon applying standard histological techniques to cell-seeded polymer scaffolds. This report describes a simple and reliable method for cryosectioning cell-polymer constructs embedded in gelatin. Solvent-soluble (PLGA) and insoluble (PGA) scaffolds were cultured in vitro with preosteoblasts, followed by histological processing with paraffin, OCT, or gelatin. Although paraffin-embedded PGA scaffolds withstood standard sectioning and rinsing steps, paraffin-embedded PLGA scaffolds were partially dissolved during the clearing step. OCT-embedded scaffolds produced sections that did not adhere well to slides, and most of the sample was lost during rinsing steps. In contrast, gelatin-embedded scaffolds exhibited adequate structural integrity during cryosectioning, adhered well to the slides, retained the actual polymer morphology, and exhibited compatibility with common stains.

The effect of pH on the structural evolution of accelerated biomimetic apatite.

The classic biomimetic apatite coating process can be accelerated by first immersing substrates into concentrated simulated body fluid, 5x SBF (SBF1), at 37 degrees C, to form an initial coating of precursor apatite spheres, and subsequently transferring to a second 5x SBF (SBF2) solution which is devoid of crystal growth inhibitors to promote phase transformation of SBF1-derived precursor apatite spheres into final crystalline apatite plates. Since SBF1 governs the formation kinetics and composition of the initial precursor spheres, we hypothesized that the pH of the SBF1 solution will also influence the final structure of the SBF2-derived crystalline apatite. To test this hypothesis, polystyrene substrates were immersed into SBF1 with different pH (5.8 or 6.5), and then immersed into the identical SBF2 (pH=6.0). The resultant apatites exhibited similar 2 theta XRD peaks; FTIR spectra in terms of hydroxyl, phosphate and carbonate groups; and Ca/P atomic ratio (1.42 for SBF1(5.8) apatite; 1.48 for SBF1(6.5) apatite). SEM, TEM and electron diffraction show that while SBF1(6.5) (pH 6.5) precursor spheres transform into larger, single crystals plates, SBF1(5.8) (pH 5.8) precursor spheres developed minute, polycrystalline plate-like structures over predominantly spherical precursor substrate.

DAZL limits pluripotency, differentiation, and apoptosis in developing primordial germ cells.

The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.

Murine embryonic stem cell derivation, in vitro pluripotency characterization, and in vivo teratoma formation.

The derivation of embryonic stem (ES) cells represents one of the most important breakthroughs in mammalian developmental biology. In addition to their utility in a wide array of in vitro studies, ES cells are also one of the most useful starting materials for the generation of mutants by homologous recombination in mice (Thomson and Solter, 1988). When ES cells are injected into host blastocysts and transferred to the uterus of a pseudo-pregnant mouse, they can contribute to different types of tissues in chimeric mice, including the germ line (Bradley et al., 1984). Hundreds of genes have been studied through genetic manipulation of ES cells to model human genetic diseases. In this unit, the ES cell lines are derived from the 129SvEv mice strain, which has a high probability of promoting germ line transmission. Procedures for validating and characterizing ES cell pluripotency are also described in detail.

Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation.

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

Nell-1-induced bone regeneration in calvarial defects.

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.