Contribution of human hematopoietic stem cells to liver repair

Immune-deficient mouse models of liver damage allow examination of human stem cell migration to sites of damage and subsequent contribution to repair and survival. In our studies, in the absence of a selective advantage, transplanted human stem cells from adult sources did not robustly become hepatocytes, although some level of fusion or hepatic differentiation was documented. However, injected stem cells did home to the injured liver tissue and release paracrine factors that hastened endogenous repair and enhanced survival. There were significantly higher levels of survival in mice with a toxic liver insult that had been transplanted with human stem cells but not in those transplanted with committed progenitors. Transplantation of autologous adult stem cells without conditioning is a relatively safe therapy. Adult stem cells are known to secrete bioactive factors that suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate recruitment, retention, mitosis, and differentiation of tissue-residing stem cells. These paracrine effects are distinct from the direct differentiation of stem cells to repair tissue. In patients at high risk while waiting for a liver transplant, autologous stem cell therapy could be considered, as it could delay the decline in liver function

Differentially expressed microRNAs in chondrocytes from distinct regions of developing human cartilage.

There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54-56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair

Contribution of human hematopoietic stem cells to liver repair

Immune-deficient mouse models of liver damage allow examination of human stem cell migration to sites of damage and subsequent contribution to repair and survival. In our studies, in the absence of a selective advantage, transplanted human stem cells from adult sources did not robustly become hepatocytes, although some level of fusion or hepatic differentiation was documented. However, injected stem cells did home to the injured liver tissue and release paracrine factors that hastened endogenous repair and enhanced survival. There were significantly higher levels of survival in mice with a toxic liver insult that had been transplanted with human stem cells but not in those transplanted with committed progenitors. Transplantation of autologous adult stem cells without conditioning is a relatively safe therapy. Adult stem cells are known to secrete bioactive factors that suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate recruitment, retention, mitosis, and differentiation of tissue-residing stem cells. These paracrine effects are distinct from the direct differentiation of stem cells to repair tissue. In patients at high risk while waiting for a liver transplant, autologous stem cell therapy could be considered, as it could delay the decline in liver function

Differentially-expressed miRNAs between differentiated chondrocytes (DC) and hypertrophic chondrocytes (HYP) from gestational day 54–56 human embryonic cartilage tissue.

<p>Fold change (f.c.) expression of miRNAs between cells of DC and HYP regions are shown. The score (d) and q-values for each differentially-expressed miRNA are shown based on SAM analysis (FDR≤5%; n = 8–9).</p

Differentially-expressed miRNAs between precursor chondrocytes (PC) and hypertrophic chondrocytes (HYP) from gestational day 54–56 human embryonic cartilage tissue.

<p>Fold change (f.c.) expression of miRNAs between cells of PC and HYP regions are shown. The score (d) and q-values for each differentially-expressed miRNA are shown based on SAM analysis (FDR≤5%; n = 8–9).</p

Enriched pathways of predicted genes targeted by differentially expressed miRNAs (PC

<p>Enriched pathways of predicted genes targeted by differentially expressed miRNAs (PC</p

Safranin-O-stained tissue section of a human embryonic developing proximal tibia (gestational day 54).

<p>Red-orange staining represents proteoglycans in the developing cartilage extracellular matrix. This stage of development is prior to endochondral ossification. Chondrocytes at various stages of differentiation are present at this stage: precursor chondrocytes (<b>PC</b>) are found at the most proximal end of the developing tibia as well as in the surrounding perichondrium; differentiated chondrocytes (<b>DC</b>) are located further down the developing limb and are distinguishable by their cuboidal or flattened phenotype, depending on their location; hypertrophic chondrocytes (<b>HYP</b>) are terminally-differentiated cells easily distinguished by their increased size. Also shown in this image are the developing femoral condyles (fc) of the distal femur. Scale bar = 250 µm.</p

Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC

<p>Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC</p

Top 30 most abundantly expressed miRNAs in precursor, differentiated and hypertrophic chondrocytes from gestational day 54–56 human embryonic cartilage tissue.

<p>Highly expressed miRNAs were identified according to their average (2^-ΔCt) values. Delta (Δ) Ct value for each miRNA was calculated by subtracting the Ct value of endogenous control, RNU44, from the Ct value of the specific miRNA. Expression level average (2^−ΔCt) in a region reflects the average of 2^−ΔCt values across all samples in that region.</p

Immunofluorescence staining of different collagen types in a human developing embryonic proximal tibia (gestational day 54).

<p>(<b>A</b>) Localization of the embryonic isoform of type II procollagen (type IIA) in the extracellular matrix (ECM). The anti-IIA antibody recognizes the exon 2-encoded cysteine-rich domain present in the amino propeptide of type IIA procollagen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075012#pone.0075012-Oganesian1" target="_blank">[43]</a>. These IIA isoforms are generated predominantly by progenitor chondrocytes seen at the periphery and most proximal area of the developing tibia. Some expression of IIA procollagen has been reported in the pre-hypertrophic and hypertrophic region of developing cartilage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075012#pone.0075012-Zhu1" target="_blank">[58]</a> and is also shown here. (<b>B</b>) Type I collagen staining is restricted to the areas of precursor chondrocytes and cells of the perichondrium/periosteum. (<b>C</b>) Type II collagen staining patterns (i.e. the processed triple helical domain of type II collagen) is present throughout the entire developing limb. (<b>D</b>) Collagen X staining is restricted to the ECM containing hypertrophic chondrocytes. Cell nuclei are visualized in blue by DAPI staining. Scale bars = 100 µm. Immunofluorescent images are representative of three independent experiments using gestational day 54 tissue sections from different embryos.</p