Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity was confirmed by 31 P-NMR. In this study, recombinant PP13/ galectin-13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin-13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin-13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI-TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin-13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin-like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin-13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin-13 may have special haemostatic and immunobiological functions at the lining of the common feto-maternal blood-spaces or developmental role in the placenta.Keywords: brush border membrane; carbohydrate binding; galectin; lysophospholipase; placental protein.Placental protein 13 (PP13) is a member of the group of the so-called Ôpregnancy-related proteinsÕ [1] that might be highly expressed in placenta and some maternal/fetal tissues during pregnancy. The structural and functional characteristics of these proteins and their possible role in placental development and regulation pathways are receiving increased interest at present. PP13 was first isolated from human placenta and characterized by Bohn et al. in 1983. It was found to be comprised of two identical 16 kDa subunits held together by disulfide bonds, and to have the lowest carbohydrate content (0.6%) of any known placental proteins [2]. Later, cloning of PP13 was performed in parallel by two research groups [3,4], and its sequence was deposited separ...

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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 use of UCB for transplantation in adults is limited due to the low cell number in each unit. Engraftment rates are lower and the time for neutrophil and platelet recovery is longer following CBT compared to bone marrow (BM) transplants. Co-transplantation of human hematopoietic stem/progenitor cells (HSC) with mesenchymal stromal cells in an irradiated NOD/SCID mice model has been demonstrated to promote HSC engraftment. Using PluriX technology, we expanded human placental derived MSC on 3D-carriers in a bioreactor (PLX-I) and evaluated its potential to promote UCB engraftment in the NOD/SCID mice model. Flow cytometer analysis detected high rates of expression of CD105, CD73, CD90 and CD29 and lacked expression of CD45, CD34 and CD19 surface markers in the PLX-I cells. PLX-I cells were capable of differentiation into bone, adipose tissue and cartilage under specific inductive conditions. Additionally, they possess both immune privileged and immune suppressive characteristics in a mixed lymphocyte reaction (MLR) assay. In this study 60 – 100x103 hUCB derived CD34+ cells were injected into the tail vein of 7–8 week old NOD/SCID mice along with 0.5x106 PLX-I cells. Human cell engraftment was tested in both sublethally irradiated (350 rad) and chemotherapy (50 mg/kg busulfan) treated NOD/SCID mice. Following 5–6 weeks, mice BM FACS analysis showed a significant increase in % hCD45+ rate in mice transplanted with PLX-I cells compared to mice transplanted with CD34+ cells alone: 13.6 vs. 31.7, p=0.01, (n=6) in the irradiation setting and 28.8 vs 6.3, p<0.05 (n=7) in the chemotherapy setting. These preclinical results demonstrate the potential of human placental-derived MSCs, grown as a 3D culture (PLX-I), to promote hUCB CD34+ cell engraftment in BM. Co-transplantation of PLX-I may be considered for improving the delayed engraftment using CB as the source of HSC.

In situ, HSCs are intimately associated with discrete spatially organized niches within the bone marrow, which are part of the “hematopoietic inductive microenvironment” (HIM). The HIM provides a range of molecular signals that collectively control HSC differentiation and self-renewal. This process is mediated via cell-cell and cell-ECM molecular contacts or through specific factors synthesized and secreted by mesenchymal stromal cells (MSC). Current strategies aimed at ex vivo expansion of transplantable HSC have so far been met with limited success. Most attempts to expand HSC ex-vivo are based on using monolayers of MSC as a supportive tier or growth media supplemented with blend of cytokines. failure to support long-term maintenance and expansion of human HSC on MSC monolayers could be associated with inadequate physical architecture of the culture systems which does not reflect the natural 3-D growth conditions present within the BM-HIM. The other method based on supporting the Hematopoietic cells proliferation by a blend of cytokines, is proven routine but post transplantation marrow replenishment is currently unsatisfactory and chromosomal epigenetic modifications may be introduced into progeny cells. Using the PluriX™ bioreactor, we had previously demonstrated that spatial co-cultures of MSC and HSCs provide an efficient system for the expansion of HSCs from CB CD34+ selected cells in cytokine-free environment. Over the past years, attempts to expand HSCs mostly employed CD34+ selected cells. However, these cells may not represent the earliest HSCs and the immunomagnetic selection protocols are costly, time consuming and associated with great loss of source cells. A theoretical approach to overcome both hurdles is by using non-selected MNC as the founding population of HSC. Following our ability to expand HSCs from CD34+ selected cells, we now demonstrate the capacity of the 3-D HSC MSCs co-culture system within the PluriX™ bioreactor system to expand HSCs using MNC as HSCs source. Primary Human marrow-derived MSCs were grown on 3-D carriers within the PluriX™bioreactor system. When the MSCs cultures reached high density (3*106–8*106 cells/ml) CD34+ selected cells or MNC were plated onto them. Within 7–14 days, the population of CD34+ and CD34+CD38− cell were expanded irrespective of the HSCs founding source. However, during this period of time the absolute expansion magnitude was greater when MNC rather than CD34+ selected cells were used to drive the process. The enrichment rate of CD34+cell from MNC and CD34+ selected cells was 20–100 vs. 5–20 fold, respectively. Under same conditions, the enrichment of the earlier CD34+CD38− cells was 40–50 vs. up to 10 fold for the same cell populations. In-conclusion, the spatial co-cultures of MSC and HSC within the PluriX™ bioreactor have been shown to form a potent HSCs expansion system in non-supplemented cytokine environment. The capacity of this practice is improved when MNCs, instead of CD34+ selected cells, serve as the originating population for HSCs. It is conceivable that the efficiency of this system is based upon selective adherence between HSCs and the hematopoietic niches within the MSCs 3-D cultures. As such, upscale of the current system could become attractive method for HSCs expansion.