Cecilia Götherström

Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells

Adult bone-marrow-derived mesenchymal stem cells are immunosuppressive and prolong the rejection of mismatched skin grafts in animals. We transplanted haploidentical mesenchymal stem cells in a patient with severe treatment-resistant grade IV acute graft-versus-host disease of the gut and liver. Clinical response was striking. The patient is now well after 1 year. We postulate that mesenchymal stem cells have a potent immunosuppressive effect in vivo.

Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta

Background. Mesenchymal stem cells (MSC) are progenitors of mesenchymal tissues such as bone, cartilage, and adipose. Adult human leukocyte antigen (HLA)-matched MSC have been used in cellular therapies of bone disorders such as osteogenesis imperfecta, with promising results. Methods. A female fetus with multiple intrauterine fractures, diagnosed as severe osteogenesis imperfecta, underwent transplantation with allogeneic HLA-mismatched male fetal MSC in the 32nd week of gestation. Engraftment analyses of donor cells, immunologic reaction against donor cells, and the well-being of the patient were assessed. Results. At 9 months of age, on slides stained for osteocalcin or osteopontin, a centromeric XY-specific probe revealed 0.3% of XY-positive cells in a bone biopsy specimen. Whole Y genome fluorescent in situ hybridization staining showed a median of 7.4% Y-positive cells (range, 6.8%–16.6%). Bone histology showed regularly arranged and configurated bone trabeculae. Patient lymphocyte proliferation against donor MSC was not observed in co-culture experiments performed in vitro after MSC injection. Complementary bisphosphonate treatment was begun at 4 months. During the first 2 years of life, three fractures were noted. At 2 years of corrected age, psychomotor development was normal and growth followed the same channel, −5 SD. Conclusions. The authors’ findings show that allogeneic fetal MSC can engraft and differentiate into bone in a human fetus even when the recipient is immunocompetent and HLA-incompatible.

Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC.

The biological properties of stem cells are key to the success of cell therapy, for which MSC are promising candidates. Although most therapeutic applications to date have used adult bone marrow MSC, increasing evidence suggests that MSC from neonatal and mid‐gestational fetal tissues are more plastic and grow faster. Fetal stem cells have been isolated earlier in development, from first‐trimester blood and hemopoietic organs, raising the question of whether they are biologically closer to embryonic stem cells and thus have advantages over adult bone marrow MSC. In this study, we show that human first‐trimester fetal blood, liver, and bone marrow MSC but not adult MSC express the pluripotency stem cell markers Oct‐4, Nanog, Rex‐1, SSEA‐3, SSEA‐4, Tra‐1‐60, and Tra‐1‐81. In addition, fetal MSC, irrespective of source, had longer telomeres (p < .001), had greater telomerase activity (p < .01), and expressed more human telomerase reverse transcriptase (p < .01). Fetal MSC were also more readily expandable and senesced later in culture than their adult counterparts (p < .01). Compared with adult MSC, first‐trimester fetal tissues constitute a source of MSC with characteristics that appear advantageous for cell therapy.

Mesenchymal Stem Cells Inhibit the Expression of CD25 (Interleukin-2 Receptor) and CD38 on Phytohaemagglutinin-Activated Lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)‐stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA‐stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA‐stimulated lymphocytes dose‐dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor‐β (TGF‐β). Stromal‐cell‐derived factor‐1 (SDF‐1) is not expressed on the cell surface. A recent report suggested that T‐cell suppression by MSC is mediated by HGF and TGF‐β. MSC suppression was not restored by the addition of neutralizing antibodies against SDF‐1, OPG, HGF or TGF‐β, alone or in combination. Addition of guanosine to PHA‐stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA‐activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.

No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients

Background and Objectives Mesenchymal stromal cells (MSC) may be used in cellular therapy to treat graft-versus-host-disease and autoimmune disorders, and in regenerative medicine. Preliminary data suggest limited cellular allogeneic rejection, but less is known about humoral responses. The objective of this study was to investigate whether antibodies against MSC were present after hematopoietic stem cell transplantation (HSCT) including treatment with HLA matched or mismatched allogeneic MSC. Design and Methods Twelve patients were evaluated using flow cytometric cross matches (FCXM) and enzyme-linked immunosorbent assays. Expression of blood group antigens, regarded as alloantigens giving rise to humoral alloimmunity, on MSC were explored using flow cytometry and immunofluorescence. Results Three of 12 patients exhibited late positivity in the FCXM. In absorption studies, antibodies directed against fetal calf serum (FCS), a component of the MSC culture medium, were identified. Healthy individuals expressed varying levels of anti-FCS antibodies and the same pattern was seen in immunosuppressed HSCT patients. MSC did not express blood group antigens. The patients with positive FCXM are alive and well. Interpretation and Conclusions We have shown that immunosuppressed patients can exhibit anti-FCS antibodies, but no alloantibodies, which may bind to MSC. These antibodies seem clinically insignificant.

Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells

Summary:Adult mesenchymal stem cells (MSCs) have been suggested to decrease lymphocyte proliferation in vitro. We hypothesised that foetal MSCs (fMSCs) would have an immunosuppressive effect on allograft responses in vitro. Human MSCs were isolated and cultured from first-trimester foetal livers and characterised by flow cytometry. fMSC stained positive for CD29, CD44, CD166, CD105, SH-3 and SH-4, and negative for CD14, CD34 and CD45. When plated on adipogenic, chondrogenic and osteogenic media, fMSC differentiated into the respective cell lineage. Compared to adult MSC (aMSC), the proliferative capacity of fMSC was higher. Mitogen stimulation of PBL was inhibited by fMSC. The greatest inhibition (78%) was seen when 30 000 fMSCs were added to 150 000 lymphocytes stimulated by phytohaemagglutinin. Adult and fMSCs were added to mixed lymphocyte cultures (MLC) containing peripheral blood lymphocytes or foetal liver cells. Unlike aMSC, fMSCs did not inhibit MLC. fMSC could be culture-expanded several million folds with no loss of phenotype characteristics, which makes them ideal for ex vivo expansion. fMSC inhibit lymphocyte proliferation induced by mitogens, but not alloreactivity as measured by MLC.

Cancer/Testis Antigen Expression in Human Mesenchymal Stem Cells: Down-regulation of SSX Impairs Cell Migration and Matrix Metalloproteinase 2 Expression

Several families of genes by and large located on the X chromosome encode proteins of unspecified function. Commonly known as cancer/testis (CT) antigens, they are considered, under normal conditions, only to be expressed in cells of the germ line and placenta. CT genes are also often expressed in cancer cells, hence their classification. Here we report that their expression in normal cells is wider spread and can be observed in cells with the potential for self-renewal and pleuripotency, namely, stem cells. Several CT genes and their products, CT antigens, including SSX, NY-ESO-1, and N-RAGE, were expressed in undifferentiated mesenchymal stem cells (MSCs) and down-regulated after osteocyte and adipocyte differentiation. To elucidate the possible overlapping function played by these genes in cancer and stem cells, a comparative analysis of the localization of their proteins was made. In addition, localization relative to other MSC markers was examined. This revealed that SSX localizes in the cytoplasm and overlap occurs in regions where matrix metalloproteinase 2 (MMP2) and vimentin accumulate. Nevertheless, it was found that no protein interactions between these molecules occur. Further investigation revealed that the migration of a melanoma cell line (DFW), which expresses SSX, MMP2, and vimentin, decreases when SSX is down-regulated. This decrease in cell migration was paralleled by a reduction in MMP2 levels. Analogous to this, SSX expression is down-regulated in MSCs after differentiation; concomitantly a reduction in MMP2 levels occurs. In addition, E-cadherin expression increases, mimicking a mesenchymal epithelial transition. These results afford SSX a functional role in normal stem cell migration and suggest a potentially similar function in cancer cell metastases.

Pre- and Postnatal Transplantation of Fetal Mesenchymal Stem Cells in Osteogenesis Imperfecta: A Two-Center Experience

Osteogenesis imperfecta (OI) can be recognized prenatally with ultrasound. Transplantation of mesenchymal stem cells (MSCs) has the potential to ameliorate skeletal damage. We report the clinical course of two patients with OI who received prenatal human fetal MSC (hfMSC) transplantation and postnatal boosting with same‐donor MSCs. We have previously reported on prenatal transplantation for OI type III. This patient was retransplanted with 2.8 × 106 same‐donor MSCs per kilogram at 8 years of age, resulting in low‐level engraftment in bone and improved linear growth, mobility, and fracture incidence. An infant with an identical mutation who did not receive MSC therapy succumbed at 5 months despite postnatal bisphosphonate therapy. A second fetus with OI type IV was also transplanted with 30 × 106 hfMSCs per kilogram at 31 weeks of gestation and did not suffer any new fractures for the remainder of the pregnancy or during infancy. The patient followed her normal growth velocity until 13 months of age, at which time longitudinal length plateaued. A postnatal infusion of 10 × 106 MSCs per kilogram from the same donor was performed at 19 months of age, resulting in resumption of her growth trajectory. Neither patient demonstrated alloreactivity toward the donor hfMSCs or manifested any evidence of toxicities after transplantation. Our findings suggest that prenatal transplantation of allogeneic hfMSCs in OI appears safe and is of likely clinical benefit and that retransplantation with same‐donor cells is feasible. However, the limited experience to date means that it is not possible to be conclusive and that further studies are required.

In vitro immunologic properties of human umbilical cord perivascular cells

BACKGROUND It has been shown recently that human umbilical cord perivascular cells (HUCPVC) are bio-equivalent to bone marrow-derived mesenchymal stromal cells (BM-MSC) in their mesenchymal differentiation and marker expression. HUCPVC populations provide high yields of rapidly proliferating mesenchymal progenitor cells. The question we wished to address, in two independent laboratory studies, was whether HUCPVC exhibit a similar in vitro immunologic phenotype to that of BM-MSC. METHODS HUCPVC were isolated by physical extraction of umbilical vessels followed by enzymatic digestion of the perivascular cells, and lymphocytes were obtained from heparinized human peripheral blood. Experimental evaluations were lymphocyte proliferation in HUPCVC or BM-MSC co-cultures with peripheral blood lymphocytes (PBL), mixed lymphocyte cultures (MLC) containing BM-MSC or HUCPVC, CD25 and CD45 expression in co-cultures containing HUCPVC, and finally lymphocyte proliferation in TransWell MLC with HUCPVC. RESULTS Both HUCPVC and BM-MSC showed no significant increase in proliferation of lymphocytes when co-cultured. The addition of 10% HUCPVC or BM-MSC significantly reduced proliferation of PBL in one-way MLC. Upon inclusion of HUCPVC with activated T-cell lines, the expression of both CD25 and CD45 showed a significant decrease. HUCPVC were able to reduce lymphocyte cell numbers significantly when separated with a membrane insert. DISCUSSION HUCPVC are not alloreactive and exhibit immunosuppression in vitro. Lymphocyte activation is significantly reduced in the presence of HUCPVC, and the immunosuppressive effect of HUCPVC is due, in part, to a soluble factor. Thus HUCPVC shows a similar immunologic phenotype to BM-MSC.

Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways.

BACKGROUND AIMS Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. METHODS We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ. RESULTS Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. CONCLUSIONS Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.