Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background: Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored. Methodology/Principal Findings: ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells. Conclusions/Significance: We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/ differentiation conditions based upon the influence of microenvironment

Evolution of HIV-1 in the Gut

The high mutation rate of Human Immunodeficiency Virus (HIV) is a significant contributor to its ability to develop drug resistance. While much research has been directed towards developing new drugs and treatments in response to resistance, it is also critical to gain a better understanding of the nature of the virus’s replication. Previous studies (van Marle et al., 2007; van Marle et al., 2010) have demonstrated that viral replication and evolution are compartmentalized in different gut tissues. However, these studies focused on the reverse transcriptase (rt) region of the proviral DNA. This project examined whether the same patterns of viral evolution would be found in the nef (negative factor) encoding region. The Nef protein contributes to the infectivity and pathogenicity of the virus and is therefore under different selection pressures than reverse transcriptase. For this project, gut biopsy samples were taken from a patient cohort at the Southern Alberta HIV Clinic from 1993-1996 and again from 2007-2010. Proviral DNA was isolated and the nef region was subsequently sequenced and analyzed using Molecular Evolutionary Genetic Analysis (MEGA) software. The results indicated that evolution of the nef region over time is consistent with compartmentalization of the gut in each patient. Overall diversity of the Nef protein encoding region is similar among all tissue types. Finally, the majority of mutations suggest that HIV-1 is under neutral or purifying selection. These observations are consistent with the observations of the rt region, suggesting a similar evolutionary pattern for the nef region

Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells

Chondrogenesis is the complex process that leads to the establishment of cartilage and bone formation. Due to their ability to differentiate in vitro and mimic development, embryonic stem cells (ESCs) show great potential for investigating developmental processes. In this study, we used chondrogenic differentiation of ESCs as a model to analyze morphogenetic events during chondrogenesis.ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.We demonstrate that ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages. The study of chondrogenesis via ESC differentiation may be informative to our further understanding of skeletal growth and regeneration

<i>In vitro</i> hypertrophy and degradation of cartilage, and bone replacement.

<p>(A) The morphology of aggregate cultured at day 50. Scale bars represent 100 µm. (B) The histological section of aggregate cultured at day 50 or 60 was stained by H&E. Hypertrophic cartilage (a), Degraded cartilage (b), Degraded cartilage and bone replacement (c) and bone replacement (d). Scale bars represent 20 µm. (C) Apoptotic cells were indicated by TUNEL staining. TUNEL (a), Bright field (b). Scale bars represent 20 µm. (D)The section of bone replacement cultured at day 60. H&E (a), Alcian Blue (b), Alizarin Red S (c), Alcian Blue & Alizarin Red S (d), Methylene Blue (e). Scale bars represent 20 µm. (E) The section of bone cultured at day 100. H&E (a), Alizarin Red S (b), COL 1 (c). Scale bars represent 20 µm.</p

Condensation of differentiated ESCs.

<p>(A) The morphology of ESC chondrogenic aggregates cultured at day 5 (a) and 15 (b), Alcian Blue at day 15 (c). Scale bars represent 100 µm. (B) The ultra-structure of static suspension aggregates was analyzed by SEM on day 15 (a, b, c). (C) The H&E histological section of chondrogenic aggregate cultured at day 15 (a), Higher magnification (b), Alcian Blue (c). Scale bars represent 20 µm. (D) Expression of Sox9, Aggrecan, COL 2 and COL 10 mRNAs were analyzed by semi-quantitative RT-PCR at 0 (ESCs), 15 and 30 days of differentiation. β-actin was used as an internal control. (E) GAG content was determined. Data is expressed as mean ± SD (n = 4) per well. * Significant difference from day 0 (ESCs). P<0.05 with Student's <i>t</i>-test. (F) Calcium accumulation during differentiation was determined. Data is expressed as mean ± SD (n = 4) per well. * Significant difference from day 0 (ESCs). ^# Significant difference from day 15 P<0.05 with Student's <i>t</i>-test.</p

Micro-mass directed ESC differentiation towards the osteoblast lineage.

<p>(A) Morphological changes on day 4 of differentiation between the six media conditions tested: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f). Scale bars represent 100 µm. (B) Apoptotic cells were observed using TUNEL on day 4 of differentiation in KSR-Dex (b) and KSR-VD3 (d). The corresponding brightfield channels demonstrates cell position (a and c respectively); scale bars represent 100 µm. (C) Re-attached aggregates on day 7 show significant differences at day 7 between the six media types: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f). (D) After trypsinization on day 7 of differentiation, aggregates also show differences between the six media types: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f); scale bars represent 100 µm. (E) Expression of osteoblast-related proteins, COL 1 and osteocalcin using immunofluorescence on day 7 of differentiation: FBS-Dex (a, m), FBS-VD3 (d, p), KSR-Dex (g, s) and KSR-VD3 (j, v). Brightfield (b, e, h, k, n, g, t, w) and DAPI (c, f, i, l, o, r, u, x) are also shown; scale bars represent 50 µm. (F) Using Alizarin Red S staining, calcification with red appearance was observed in culture dish on day 7 and 14 of differentiation: FBS-basic (a, d), FBS-Dex (b, e), FBS-VD3 (c, f), KSR-basic (g, j), KSR-Dex (h, k), KSR-VD3 (i, l); scale bars represent 50 µm. (G) Calcium content per micro-mass spot (a) and normalized against DNA (b) was determined; data is expressed as mean ± SD (n = 3) per well. ^# represents a significant difference between FBS-Dex and VD3 or KSR-Dex and VD3; P<0.05 with Student's <i>t-</i>test. * represents a significant difference between FBS and KSR, P<0.05 with Student's <i>t-</i>test. (H) Expression of osteoblast-related mRNAs (Cbfa1, osteocalcin) was analyzed using real-time PCR at 0 (ESCs), 7 and 14 days. Data is expressed as means ± SD (n = 3) per lane.</p

Bone-like tissue formation <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The ultra-structure of aggregates was analyzed by SEM on day 15 of stirred suspension culture: KSR-basic (a, d), KSR-Dex (b, e), KSR-VD3 (c, f). Low magnification (a, b, c), scale bars represent 50 µm; high magnification (d, e, f), scale bars represent 10 µm. (B) Histological sections of aggregates were analyzed by hematoxylin and eosin (H&E) staining after 15 days of differentiation: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c); scale bars represent 20 µm. (C) Expression of osteoblast-related mRNAs (Cbfa1, osteocalcin) was analyzed using real-time PCR during ESC differentiation in KSR-VD3 static suspension culture. (D) Calcium content was analyzed during ESC differentiation in KSR-VD3 static suspension culture and normalized against DNA content. (E) Expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR during ESC differentiation in KSR-VD3 static suspension culture; data is expressed as means ± SD (n = 3) per lane. * represents a significant difference between two conditions tested; P<0.05 with Student's <i>t-</i>test. (F) Following 15 days of differentiation in static suspension culture, bone (a, b, e, f) and epithelium (c, d, g, h)-like tissues were observed <i>in vivo</i> upon transplantation into SCID mice: KSR-Dex (a, b, c, d) and KSR-VD3 (e, f, g, h); H&E (a, c, e, g) and Methylene Blue (b, d, f, h) staining, scale bars represent 20 µm.</p

Gene expression during chondrogenesis in suspension culture.

<p>The expression of chondrocyte and osteoblast-related genes was analyzed from 0 to 60 days of differentiation by real-time RT-PCR. Sox9 (a), Aggrecan (b), COL 2 (c), COL 10 (d), MMP13 (e), Cbfa1 (f), Osteocalcin (g). Data is expressed as means ± SD (n = 3) per lane. With a P<0.05 using the Student's <i>t</i>-test, * represents a significant difference between two.</p

Chondrocyte differentiation.

<p>(A) The ultra-structure of static suspension aggregate was analyzed by SEM at day 15. (B) The cells shed from aggregates displayed two types, round-shaped with fibrils (a) and flat fibroblastic cells (b). Scale bars represent 20 µm. (C) The round-shaped cells formed network spontaneously. Scale bars represent 50 µm. (D) The expression of chondrocyte-related proteins (Aggrecan, COL 2, COL 10) was analyzed at day 20 of differentiation by immunofluorescence (a, d, g), DAPI (b, e, h), Bright field (c, f, i). Scale bars represent 50 µm. (E) Alcian Blue stained chondrogenic cells at day 20 of differentiation (a, b). These cells were induced to form the aggregates spontaneously (c, d). Scale bars represent 50 µm. (F) The expression of chondrocyte-related mRNAs (Sox9, Aggrecan, COL 2, COL 10) and fibroblast-related mRNA (COL 1) were analyzed at day 20 by semi-quantitative RT-PCR. β-actin was used as a loading control.</p

Schematic summary of the stages of chondrogenesis of ESCs.

<p>The relative temporal aspects of five stages of chondrogenesis are denoted by cellular, extracellular and molecular events: (1) Condensation of differentiated ESCs, (2) Differentiation into chondrocytes and fibril scaffold formation, (3) ECM deposition and cartilage formation, (4) Hypertrophy and degradation of cartilage, and (5) Bone replacement. Scale bars represent 20 µm.</p