Proliferative and chondrogenic potential of mesenchymal stromal cells from pluripotent and bone marrow cells.

INTRODUCTION: Mesenchymal stromal cells (MSCs) can be derived from a wide range of fetal and adult sources including pluripotent stem cells (PSCs). The properties of PSC-derived MSCs need to be fully characterized, in order to evaluate the feasibility of their use in clinical applications. PSC-MSC proliferation and differentiation potential in comparison with bone marrow (BM)-MSCs is still under investigation. The objective of this study was to determine the proliferative and chondrogenic capabilities of both human induced pluripotent stem cell (hiPSC-) and embryonic stem cell (hESC-) derived MSCs, by comparing them with BM-MSCs. METHODS: MSCs were derived from two hiPSC lines (hiPSC-MSCs), the well characterized Hues9 hESC line (hESC-MSCs) and BM from two healthy donors (BM-MSCs). Proliferation potential was investigated using appropriate culture conditions, with serial passaging, until cells entered into senescence. Differentiation potential to cartilage was examined after in vitro chondrogenic culture conditions. RESULTS: BM-MSCs revealed a fold expansion of 1.18x105 and 2.3x105 while the two hiPSC-MSC lines and hESC-MSC showed 5.88x10¹°, 3.49x108 and 2.88x108, respectively. Under chondrogenic conditions, all MSC lines showed a degree of chondrogenesis. However, when we examined the formed chondrocyte micromasses by histological analysis of the cartilage morphology and immunohistochemistry for the chondrocyte specific markers Sox9 and Collagen II, we observed that PSC-derived MSC lines had formed pink rather than hyaline cartilage, in contrast to BM-MSCs. CONCLUSION: In conclusion, MSCs derived from both hESCs and hiPSCs had superior proliferative capacity compared to BM-MSCs, but they were inefficient in their ability to form hyaline cartilage.

Extramedullary leukemia relapse after allogeneic stem cell transplantation: a novel mechanism of immune escape?

Background: Relapse is a significant cause of treatment failure after allogeneic stem cell transplantation. In many cases relapse occurs when leukemic cells escape from immune surveillance. Methods & results: In the setting of haploidentical transplantation, immune escape is usually the result of the loss of the mismatched haplotype from leukemic cells, while downregulation of HLA-expression has been postulated as a significant cause of immune escape after transplantation with the use of HLA-matched donors. We observed that patients with acute leukemia who relapse at the time of active graft-versus-host-disease, usually develop extramedullary leukemia while they remain free of leukemia in peripheral blood and bone marrow. Conclusion: Our observation points toward a novel mechanism of immune escape which is microenvironment-specific.

Therapeutic Effects of Mesenchymal Stem Cells Derived From Bone Marrow, Umbilical Cord Blood, and Pluripotent Stem Cells in a Mouse Model of Chemically Induced Inflammatory Bowel Disease.

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases, such as necrotizing enterocolitis (NEC), gastrointestinal graft versus host disease (GVHD), and the primary acute phase of chronic inflammatory bowel disease (CIBD), exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study, we comparatively assess the efficacy of human MSCs derived from bone marrow (BM), umbilical cord blood (UCB), human embryonic stem cells (ESCs), or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs), UCB (UCB-MSCs), ESCs (ESC-MSCs), or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group, significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment.

Natural killer cell cytotoxicity is a predictor of outcome for patients with high risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia treated with azacytidine.

The aim of the present study was to identify biomarkers predictive of the outcome of patients with high-risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia (AML) treated with 5-azacytidine (AZA). We prospectively examined the association between NK-cytotoxic activity, myeloid-derived suppressor cells (MDSCs), and T-regulatory cells (Tregs) on the overall survival (OS) of patients. Patients with NK-cytotoxicity above a critical threshold had a longer duration of response and survived longer than patients with severe impairment of NK-cytotoxicity. The numbers of MDSCs, and Tregs in the PB of patients after a short exposure to AZA were not different from normal donors. In conclusion, the results of our study suggest that the therapeutic activity of AZA is at least partly mediated by an immunomodulatory effect. To our knowledge, this is the first study reported so far, that shows a positive correlation between NK cytotoxicity and OS of AZA-treated patients.

Reprogramming of bone marrow derived mesenchymal stromal cells to human induced pluripotent stem cells from pediatric patients with hematological diseases using a commercial mRNA kit.

The potential use of patient-specific induced pluripotent stem cells (hiPSCs) in the study and treatment of hematological diseases requires the setup of efficient and safe protocols for hiPSC generation. We aimed to adopt a reprogramming method for large-scale production of integration-free patient-specific hiPSC-lines in our stem cell processing laboratory, which supports a pediatric hematopoietic stem cell transplant unit located at a tertiary care children's hospital. We describe our 5-year experience in generation of hiPSC-lines from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) using synthetic mRNAs encoding reprogramming factors. We generated hiPSC-lines from pediatric patients with ß-Thalassemia, Sickle Cell Anemia, Blackfan-Diamond Anemia, Severe Aplastic Anemia, DOCK8 Immunodeficiency and 1 healthy control. After optimization of the reprogramming procedure, average reprogramming efficiency of BM-MSCs was 0.29% (range 0.25-0.4). The complete reprogramming process lasted 14-16 days. Three to five hiPSC-colonies per sample were selected, expanded to 5 culture passages and then frozen. The whole procedure took an average time of 1.8 months (range 1.6-2.2). The hiPSC-lines expressed embryonic stem cell markers and exhibited pluripotency. This mRNA reprogramming method can be applicable in a hematopoietic stem cell culture lab setting and would be useful for the clinical translation of patient-specific hiPSCs.

Generation of human ß-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with ß-thalassemia (ß-thal) with the aim to generate trangene-free ß-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in ß-thal-iPSC lines. After 10 serial passages in vitro, ß-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free ß-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Mesenchymal derivatives of genetically unstable human embryonic stem cells are maintained unstable but undergo senescence in culture as do bone marrow-derived mesenchymal stem cells.

Recurrent chromosomal alterations have been repeatedly reported in cultured human embryonic stem cells (hESCs). The effects of these alterations on the capability of pluripotent cells to differentiate and on growth potential of their specific differentiated derivatives remain unclear. Here, we report that the hESC lines HUES-7 and -9 carrying multiple chromosomal alterations produce in vitro mesenchymal stem cells (MSCs) that show progressive growth arrest and enter senescence after 15 and 16 passages, respectively. There was no difference in their proliferative potential when compared with bone marrow-derived MSCs. Array comparative genomic hybridization analysis (aCGH) of hESCs and their mesenchymal derivatives revealed no significant differences in chromosomal alterations, suggesting that genetically altered hESCs are not selected out during differentiation. Our findings indicate that genetically unstable hESCs maintain their capacity to differentiate in vitro into MSCs, which exhibit an in vitro growth pattern of normal MSCs and not that of transformed cells.