Boon Chin Heng

Directing Stem Cell Differentiation into the Chondrogenic Lineage In Vitro

A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well‐defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage‐related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.

The immunogenicity and immunomodulatory function of osteogenic cells differentiated from mesenchymal stem cells

Multipotent mesenchymal stem cells (MSC) are reported to be immunoprivileged as well as immunosuppressive. Hence, they are ideal candidates for allogeneic transplantation to induce regeneration of diseased tissues and organs. However, it is not known whether MSC would retain their immunoprivileged and immunomodulatory properties after differentiating into the local cell types of the transplantation site. This study sought to investigate this question with a novel New Zealand White rabbit osteogenesis model. Results showed that osteogenic cells differentiated from MSC (DOC) in vitro did not express the MHC class II molecule, were incapable of inducing allogeneic lymphocyte proliferation in mixed lymphocyte culture or generating CTL, were inhibitory in ongoing lymphocyte proliferation, and secreted anti-inflammatory cytokines (IL-10 and TGF-β). There was a significantly higher secretion of IL-10 by DOC than that by MSC, while there was no significant difference between the TGF-β secretion of MSC and DOC in vitro. However, after IFN-γ treatment, TGF-β secretion by DOC significantly decreased despite the increased production by MSC. Four weeks after local DOC implantation, despite MHC class II expression, second-set allogeneic skin rejection showed similar survival to first-set allogeneic skin rejection and DOC appeared to function as osteoblasts. In conclusion, DOC retained their immunoprivileged and immunomodulatory properties in vitro, but the latter was lost following transplantation.

Strategies for Directing the Differentiation of Stem Cells Into the Osteogenic Lineage In Vitro

A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well‐defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone‐related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro.

Effects of Culture Conditions and Bone Morphogenetic Protein 2 on Extent of Chondrogenesis from Human Embryonic Stem Cells

The study of human embryonic stem cells (hESCs) can provide invaluable insights into the development of numerous human cell and tissue types in vitro. In this study, we addressed the potential of hESCs to undergo chondrogenesis and demonstrated the potential of hESC‐derived embryoid bodies (EBs) to undergo a well‐defined full‐span chondrogenesis from chondrogenic induction to hypertrophic maturation. We compared chondrogenic differentiation of hESCs through EB direct‐plating outgrowth system and EB‐derived high‐density micromass systems under defined serumfree chondrogenic conditions and demonstrated that cell‐tocell contact and bone morphogenetic protein 2 (BMP2) treatment enhanced chondrocyte differentiation, resulting in the formation of cartilaginous matrix rich in collagens and proteoglycans. Provision of a high‐density three‐dimensional (3D) microenvironment at the beginning of differentiation is critical in driving chondrogenesis because increasing EB seeding numbers in the EB‐outgrowth system was unable to enhance chondrogenesis. Temporal order of chondrogenic differentiation and hypertrophic maturation indicated by the gene expression profiles of Col 1, Col 2, and Col 10, and the deposition of extracellular matrix (ECM) proteins, proteoglycans, and collagen II and X demonstrated that the in vivo progression of chondrocyte maturation is recapitulated in the hESC‐derived EB model system established in this study. Furthermore, we also showed that BMP2 can influence EB differentiation to multiple cell fates, including that of extraembryonic endodermal and mesenchymal lineages in the EB‐outgrowth system, but was more committed to driving the chondrogenic cell fate in the EB micromass system. Overall, our findings provide a potential 3D model system using hESCs to delineate gene function in lineage commitment and restriction of chondrogenesis during embryonic cartilage development.

Combined effects of TGFβ1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation

This study investigated the effects of TGFβ1, BMP2 or a combination of both on the chondrogenic differentiation of mesenchymal stem cells (MSCs) in a serum-free micromass culture system in vitro. Putative MSCs harvested from the iliac crest of 4–5 month old New Zealand White Rabbits were expanded and cultured in three-dimensional high density micromass aggregate cultures containing TGFβ1, BMP2 or a combination of both, in the absence of serum. After 14–20 days of culture, chondrogenic differentiation of the MSCs was assayed by toluidine blue staining, immunohistochemistry and semi-quantitative RT-RCR of type I collagen (CI) and type II collagen (CII). Quantitative measurements of cell proliferation and sulfated glycosaminoglycan (s-GAG) were also carried out to assess the growth rate and matrix deposition of the cultured aggregates. Both immunohistochemical staining and semi-quantitative RT-PCR showed that the combination of BMP2 and TGFβ1 resulted in a marked enhancement of collagen II synthesis, with minimal collagen I expression, which would suggest hyaline-like cartilage formation. Additionally, BMP2 and TGFβ1 had a synergistic effect on matrix proteoglycan deposition, as assessed by metachromatic toluidine blue staining. This is consistent with the quantitative measurement of glycosaminoglycans, whereby a significant increase in GAG/DNA was noted in the co-treatment group. Hence, it can be concluded that the combination of BMP2 and TGFβ1 has a synergistic effect on the differentiation of MSC into hyaline-like cartilage tissue.

Mesenchymal stem cell sheets revitalize nonviable dense grafts: implications for repair of large-bone and tendon defects.

Background. Large musculoskeletal defects are commonly reconstructed with allogeneic grafts. As cryopreserved allogeneic grafts lack viable cells, this often results in poorer clinical outcome. Current technology can not incorporate large number of cells to the dense grafts. This study aimed to investigate the feasibility of fabricating sheets of mesenchymal stem cells (MSCs) to revitalize cryopreserved grafts. Methods. Human MSCs were isolated, characterized, and cultured to form a cell sheet in the presence of ascorbic acid. Once a sheet of MSCs was obtained, it was assembled onto the demineralized bone grafts or frozen tendon grafts by a wrapping technique. Then the assembled structure was cultured for 3 weeks. The macro morphology, histology, and immunohistochemistry of the grafts were evaluated. Results. It was found that MSCs were able to form coherent cellular sheets within 3 weeks. When assembled with demineralized bone matrix, MSC sheets were similar to in situ periosteum and were able to differentiate into the osteochondral lineage. When assembled with frozen tendon graft, MSCs sheets were well-incorporated within the tissue sheath (peritenon) around the tendon, and adopted the characteristic spindle-shaped morphology of tenocyte-like cells. Conclusions. The results therefore demonstrated that MSCs sheets are easily fabricated and can maintain their differentiation potential within particular scaffolds, which would suggest a novel and convenient strategy for revitalizing large tissue grafts to improve clinical outcome.

The cryopreservation of human embryonic stem cells

hES (human embryonic stem) cells hold tremendous potential in the newly emerging field of regenerative medicine, in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening. However, an essential prerequisite for the future widespread application of hES cells are the development of efficient cryopreservation protocols to facilitate their storage and transportation. This review summarizes the current state of progress in the field of hES cell cryopreservation, by critically examining and comparing the various cryopreservation protocols that have been developed. These can be classified into two categories: (1) conventional slow‐cooling protocols and (2) vitrification protocols. Previously, the application of slow‐cooling cryopreservation protocols to freely‐suspended hES cell clumps yielded extremely dismal results. However, a recent study demonstrated that post‐thaw survivability was markedly improved when slow‐cooling protocols were applied instead to adherent hES colonies. Vitrification protocols have been shown to be much better than the standard slow‐cooling protocol for the cryopreservation of freely suspended hES cell clumps. However, no study has yet attempted to apply vitrification protocols to adherent hES colonies. It must be noted that vitrification protocols are extremely labour‐intensive and tedious to perform manually. Additionally, the use of cryostraws in vitrification protocols is unsuited for handling bulk quantities of hES cells, in addition to posing serious technical difficulties in developing machine automation for cryopreservation. These are some of the major challenges that have to be overcome if further progress is to be made in this field.

Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems

The objective of this study was to compare the osteogenic potential of human embryonic stem cells (hESCs) within two‐ and three‐dimensional (2D and 3D) culture systems. hESCs of the H1 line (Wicell Inc., Madison, Wisc., USA) were induced to form embryoid bodies (EBs) through 5 days of suspension culture within non‐adherent culture dishes. Following enzymatic dissociation, the EB‐derived single cells were seeded on either novel 3D porous PLGA scaffolds or 2D culture dishes with the same total cell number. Osteogenic differentiation was induced through culture media supplemented with dexamethasone, L‐ascorbic acid and β‐glycerophosphate. After 3 weeks of in vitro culture, quantitative and qualitative assays of osteogenic differentiation were conducted. Osteocalcin secretion and alkaline phosphatase (AP) activities were detected at significantly higher levels within 3D culture compared with the 2D system. Subsequently, the cell‐scaffold constructs were implanted in iliac crest defects of immunosuppressed rabbits. After 4 weeks, the constructs were subsequently explanted and characterized by histology and X‐ray analysis. Formation of new bone was detected within and around the implanted scaffolds. The results demonstrate that the osteogenic differentiation of human embryonic stem cells is enhanced in a 3D culture system compared to a 2D culture environment. Upon implantation in situ, the differentiating human embryonic stem cells can contribute positively to the repair and regeneration of bone defects.

Mohawk Promotes the Tenogenesis of Mesenchymal Stem Cells Through Activation of the TGFβ Signaling Pathway

The transcription factor Mohawk (Mkx) is expressed in developing tendons and is an important regulator of tenogenic differentiation. However, the exact roles of Mkx in tendinopathy and tendon repair remain unclear. Using gene expression Omnibus datasets and immunofluorescence assays, we found that Mkx expression level was dramatically lower in human tendinopathy tissue and it is activated at specific stages of tendon development. In mesenchymal stem cells (MSCs), ectopic Mkx expression strikingly promoted tenogenesis more efficiently than Scleraxis (Scx), a well‐known master transcription factor of tendon. Significantly higher levels of tenogenic gene expression and collagen fibril growth were observed with Mkx versus Scx. Interestingly, it was observed that Mkx dramatically upregulated Scx through binding to the Tgfb2 promoter. Additionally, the transplantation of Mkx‐expressing‐MSC sheets promoted tendon repair in a mouse model of Achilles‐tendon defect. Taken together, these data shed light on previously unrecognized roles of Mkx in tendinopathy, tenogenesis, and tendon repair as well as in regulating the TGFβ pathway. Stem Cells 2015;33:443–455

Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate.

Embryo quality is strongly dependent on the in-vitro culture environment. Conventionally, IVF/intracytoplasmic sperm injection (ICSI) embryos are examined microscopically every morning (from day 1 to day 6) to assess fertilization, cleavage and embryo quality. Consequently, the frequent exposure to non-optimal conditions outside the incubator may adversely affect embryonic viability and quality. Hence, this study investigated whether reduction of observation frequency outside the incubator can enhance blastocyst formation rate. A total of 285 IVF/ICSI cycles were divided into two groups. Embryos in the control group (103 cycles) were assessed out-of-incubator every day after insemination (day 1 to day 6; six times). In the experimental group (182 cycles), embryos were assessed four times, on days 1, 3, 5 and 6. The total blastocyst formation rate, day-5 blastocyst formation rate, proportion of good blastocysts and number of cryopreserved blastocysts per patient were significantly lower for the control group compared with the experimental group (42.5%, 31.4%, 50.7%, 1.72+/-1.55 versus 52.6%, 40.7%, 60.1%, 2.64+/-2.59, respectively, P<0.05); although there were no significant differences in the proportions of good embryos on day 3, blastocyst formation rate on day 6, clinical pregnancy rate and implantation rate. Hence, reduction of the observation frequency of embryos outside the incubator can enhance embryo quality and blastocyst formation rate.