Autologous cell-coated particles for the treatment of segmental bone defects-a new cell therapy approach.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are one of the most potent adult stem cells, capable of differentiating into bone, cartilage, adipose, muscle, and others. An innovative autologous AT-MSC-derived cell-based product (BonoFill-II) for bone tissue regeneration was developed to be suited as a bone graft for segmental bone defects. METHODS: BonoFill-II was transplanted into 8 sheep with 3.2-cm full cortex segmental defect formed in the tibia. Bone regeneration was followed by X-ray radiographs for 12 weeks. At experiment termination, the healed tibia bones were analyzed by computed tomography, histology, and mechanical tests. RESULTS: Our results indicate that one dose of BonoFill-II injectable formula led to an extensive bone growth within the transplantation site and to a complete closure of the critical gap in the sheep's tibia in a relatively short time (8-12 weeks), with no inflammation and no other signs of graft rejection. This new and innovative product opens new prospects for the treatment of long bone defects. CONCLUSIONS: Injection of BonoFill-II (an innovative autologous cell therapy product for bone tissue regeneration) into a critical size segmental defect model (3.2 cm), generated in the sheep tibia, achieved full bridging of the gap in an extremely short period (8-12 weeks).

The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

Differentiation of human embryonic stem cells into insulin-producing clusters.

Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing beta cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin-producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of beta cells for transplantation. We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells. The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers. Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells. Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.