New therapeutic approaches in pediatric diseases: Mesenchymal stromal cell and mesenchymal stromal cell-derived extracellular vesicles as new drugs

Mesenchymal Stromal Cell (MSC) clinical applications have been widely reported and their therapeutic potential has been documented in several diseases. MSCs can be isolated from several human tissues and easily expanded in vitro, they are able to differentiate in a variety of cell lineages, and they are known to interact with most immunological cells, showing immunosuppressive and tissue repair properties. Their therapeutic efficacy is closely associated with the release of bioactive molecules, namely Extracellular Vesicles (EVs), effective as their parental cells. EVs isolated from MSCs act by fusing with target cell membrane and releasing their content, showing a great potential for the treatment of injured tissues and organs, and for the modulation of the host immune system. EV-based therapies provide, as major advantages, the possibility to cross the epithelium and blood barrier and their activity is not influenced by the surrounding environment. In the present review, we deal with pre-clinical reports and clinical trials to provide data in support of MSC and EV clinical efficacy with particular focus on neonatal and pediatric diseases. Considering pre-clinical and clinical data so far available, it is likely that cell-based and cell-free therapies could become an important therapeutic approach for the treatment of several pediatric diseases

Proliferation Pattern of Pediatric Tumor-Derived Mesenchymal Stromal Cells and Role in Cancer Dormancy: A Perspective of Study for Surgical Strategy

: The explanation for cancer recurrence still remains to be fully elucidated. Moreover, tumor dormancy, which is a process whereby cells enter reversible G0 cell cycle arrest, appears to be a critical step in this phenomenon. We evaluated the cell cycle proliferation pattern in pediatric tumor-derived mesenchymal stromal cells (MSCs), in order to provide a better understanding of the complex mechanisms underlying cancer dormancy. Specimens were obtained from 14 pediatric patients diagnosed with solid tumors and submitted to surgery. Morphology, phenotype, differentiation, immunological capacity, and proliferative growth of tumor MSCs were studied. Flow cytometric analysis was performed to evaluate the cell percentage of each cell cycle phase. Healthy donor bone marrow-derived mesenchymal stromal cells (BM-MSCs) were employed as controls. It was noted that the DNA profile of proliferating BM-MSC was different from that of tumor MSCs. All BM-MSCs expressed the typical DNA profile of proliferating cells, while in all tumor MSC samples, ≥70% of the cells were detected in the G0/G1 phase. In particular, seven tumor MSC samples displayed intermediate cell cycle behavior, and the other seven tumor MSC samples exhibited a slow cell cycle. The increased number of tumor MSCs in the G0-G1 phase compared with BM-MSCs supports a role for quiescent MSCs in tumor dormancy regulation. Understanding the mechanisms that promote dormant cell cycle arrest is essential in identifying predictive markers of recurrence and to promote a dedicated surgical planning

CPAM type 2-derived mesenchymal stem cells: Malignancy risk study in a 14-month-old boy

INTRODUCTION: The association between congenital pulmonary airway malformations (CPAM) and malignancy is reported in the literature. Interactions between the tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. We characterized MSCs isolated from CPAM lesions in order to define potential malignancy risks. METHODS: CPAM II pulmonary tissue was used for MSC expansion; a \u201chealthy\u201d lung section from the same child was used as a comparator. Morphology, immunophenotype, differentiation and immunological capacity, proliferative growth, gene signature telomerase activity, and in vivo tumorigenicity in nude mice were evaluated. RESULTS: MSCs were successfully isolated and propagated from CPAM tissue. CPAM\u2010MSCs presented the typical MSC morphology and phenotype, while exhibiting high proliferative capacity, reaching confluence at a median time of 5 days as well as differentiation capabilities. CPAM\u2010MSCs at early passages were not neoplastic and chromosomally normal, even though unbalanced chromosomal rearrangements were noted by molecular karyotype. CCONCLUSIONS: CPAM\u2010MSCs exhibited specific features similar to tumor derived MSCs. Whilst there was no evidence of malignant transformation in the cystic tissue, our results provide evidence that this abnormal tissue has malignant potential. MSCs are considered important players in the tumor microenvironment and they have been closely linked to regulation of tumor survival, growth, and progression. Thus, early lesion resection also in asymptomatic patients might be indicated to exclude that the microenvironment may be potentially permissive to cancer development

MKRN3 and KISS1R mutations in precocious and early puberty

Background: Pubertal timing is known to be influenced by interactions among various genetic, nutritional, environmental and socio-economic factors, although the ultimate mechanisms underlying the increase in pulsatile GnRH secretion at puberty have yet to be fully elucidated. The aim of our research was to verify the role of KISSR1 (previously named GPR54) and MKRN3 genes on pubertal timing. Methods: We analyzed the DNA sequence of these genes in 13 girls affected by central precocious puberty (CPP) who showed onset of puberty before 8 years of age, and in 6 girls affected by early puberty (EP) between 8 and 10 years of age. Results: Direct sequencing of the KISS1R (GPR54) gene revealed two SNPs. One SNP is a missense variant (rs 350,132) that has been previously reported in connection to CPP in Korean girls. The other variant that we found in the GPR54 gene (rs764046557) was a missense SNP located in exon 5 at position 209 of the aminoacid. We identified this variant in only one CPP patient. Automatic sequencing of MKRN3 in all patients revealed three variants in eight subjects. In 6 out of 19 (31.5%) patients (3/13 CPP patients and 3/6 EP patients) we found the synonymous variant c.663C > T (rs2239669). Another synonymous variant (rs140467331) was found in one of our CPP patients, as well as one missense variant (rs760981395) in another CPP patient. Conclusion: In conclusion, we identified sequence variations of the KISS1R and MKRN3 genes, two of the most frequent genetic causes of ICPP. Our results suggest that these variants might be inducible factors in the pathogenesis of CPP

Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse

Use of alternative donors/sources of hematopoietic stem cells (HSC), such as cord blood (CB) or HLA-haploidentical (Haplo)-related donors, is associated with a significant delay in immune reconstitution after transplantation. Long-term T-cell immune reconstitution largely relies on the generation of new T cells in the recipient thymus, which can be evaluated through signal joint (sj) and beta T-cell-Receptor Excision Circles (TREC) quantification. We studied two groups of 33 and 24 children receiving, respectively, HSC Transplantation (HSCT) from an HLA-haploidentical family donor or an unrelated CB donor, for both malignant (46) and non-malignant disorders (11). Relative and absolute sj and beta-TREC values indicated comparable thymic function reconstitution at 3 and 6 months after the allograft in both groups. Compared to children with non-malignant disorders, those with hematological malignancies had significantly lower pre-transplantation TREC counts. Patients who relapsed after HSCT had a significantly less efficient thymic function both before and 6 months after HSCT with especially low beta-TREC values, this finding suggesting an impact of early intra-thymic T-cell differentiation on the occurrence of leukemia relapse

Pre- and post-transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre-HSCT and during the first and third trimesters after HSCT (post-HSCT1 and post-HSCT3). The overall event-free survival (EFS) was 50%. The cumulative incidence of relapse and non-relapse mortality was 41% and 9%. Any degree of detectable pre-HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 x 10-3 or 651 x 10-3, respectively, versus 73% in MRD-negative patients (P < 0001). This effect was maintained in different disease remissions, but low-level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post-HSCT1 and post- HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre-emptive immuno-therapy

Mesenchymal Stromal Cell on Liver Decellularised Extracellular Matrix for Tissue Engineering

Background: In end-stage chronic liver disease, transplantation represents the only curative option. However, the shortage of donors results in the death of many patients. To overcome this gap, it is mandatory to develop new therapeutic options. In the present study, we decellularised pig livers and reseeded them with allogeneic porcine mesenchymal stromal cells (pMSCs) to understand whether extracellular matrix (ECM) can influence and/or promote differentiation into hepatocyte-like cells (HLCs). Methods: After decellularisation with SDS, the integrity of ECM-scaffolds was examined by histological staining, immunofluorescence and scanning electron microscope. DNA quantification was used to assess decellularisation. pMSCs were plated on scaffolds by static seeding and maintained in in vitro culture for 21 days. At 3, 7, 14 and 21 days, seeded ECM scaffolds were evaluated for cellular adhesion and growth. Moreover, the expression of specific hepatic genes was performed by RT-PCR. Results: The applied decellularisation/recellularisation protocol was effective. The number of seeded pMSCs increased over the culture time points. Gene expression analysis of seeded pMSCs displayed a weak induction due to ECM towards HLCs. Conclusions: These results suggest that ECM may address pMSCs to differentiate in hepatocyte-like cells. However, only contact with liver-ECM is not enough to induce complete differentiation.</p

Phenotypical, Functional and Genetic Characterization of Mesenchymal Stem Cells Derived from the Spleen of Patients with Myelofibrosis.

Splenic extramedullary hematopoiesis is a major clinico-pathological feature of patients with myelofibrosis. As in the bone marrow (BM), hematopoiesis in the spleen occurs thank to the interplay of hematopoietic progenitor cells with the microenvironment, which provides the regulatory mechanism for their differentiation, proliferation and trafficking. Among other components, such as vessels and extra-cellular matrix proteins, this microenvironment encompasses different types of accessory cells, including mesenchymal stromal cells (MSCs). We have recently reported that MSCs from the BM of patients with myelofibrosis harbor genetics abnormalities and display an altered functional activity, suggesting that a primary MSC defect may either lead to or favor the pathogenesis of the disease. Here, we describe the phenotypical, functional, and genetic profile of MSCs isolated from the spleen of 23 patients with myelofibrosis, who underwent splenectomy for anemia and/or for excessive size of the spleen, and compare them to splenic mesenchymal stromal cells (s-MSCs) from 7 healthy subjects (HSs) who were splenectomized following traumatic lesion. The study was approved by the institutional review board of IRCSS Policlinico San Matteo Foundation; patients and HSs gave written informed consent for participating to the study. Mononuclear cells (MNCs) were obtained by dissociation of small spleen fragments by means of the GentleMacs Dissociator device (Miltenyi Biotech, Germany), and s-MSCs were isolated and expanded according to the standard procedures used for BM-MSCs. S-MSCs were obtained in 9/23 patients and in 3/7 HSs and displayed no significant differences for morphology and differentiation ability into adipocytic and osteoblastic lineages. However, the clonogenic efficiency of s-MSCs from patients with myelofibrosis was statistically higher than that of HSs (0.07 colonies/106 MNCs, range 0.03-0.01, vs 0.03/106 MNCs, range 0.03-0.04, respectively; p=0.048), whereas doubling time and time to senescence were not statistically different. Flow cytometric assessment of standard surface antigens (CD13, CD14, CD34, CD45, CD73, CD90, CD105) confirmed the mesenchymal nature of the cells grown in the cultures, and was similar between patients’ and HSs’ s-MSCs. When nestin expression was determined, no significant differences in the frequency of MSCs expressing this antigen was observed; however, nestin Mean Fluorescence Intensity (MFI) of patients’ s-MSCs was significantly lower than that of s-MSCs from HSs (22, range 6-45, vs 97, range 65-100, respectively; p=0.035). Patients’ s-MSCs also displayed a reduced capacity to sustain long term hematopoiesis in vitro in a classical Long Term Culture-Initiating Cell assay. However, when normal cord blood-derived CD34+ cells were co-cultured onto patients’ s-MSCs in a transwell system for 13 days, the output of CD41+ megakaryocytic cells increased with respect to culture where CD34+ cells were plated onto HSs' s-MSCs [21,5% vs 14,2% w/o recombinant human thrombopoietin (rhTPO), respectively, p=0,043; 60,2% vs 33,6% with rhTPO, respectively, p=0,01] at detriment of CD33+ cells (41,5% vs 48,6% w/o rhTPO, respectively, p=0,049; 10,4% vs 29,4% with rhTPO, respectively, p=0,012]. Finally, an abnormal karyotype [46XXt(5;17)(4-12)] was detected in 1 out of 18 metaphases of 1 out of 3 patient s-MSCs, while a normal karyotype was always observed in 2 out of 2 HSs’ s-MSC. This extensive characterization of s-MSCs shows that s-MSCs of patients with myelofibrosis display functional and genetic abnormalities compared to those isolated from HSs. The low level of nestin expression suggests that the hematopoietic niche of the spleen of patients with myelofibrosis can be defective and responsible for the increased trafficking of CD34+ cells that is observed in these patients, whereas the increased differentiation into the megakaryocytic lineage indicates a role of the splenic niche in leading hematopoiesis toward a pathological profile. All together, our data suggest that s-MSCs play a role in the pathogenesis of myelofibrosis and could be, therefore, a potential target for the treatment of the disease