Rosaria Giordano

Oct-4 Expression in Adult Human Differentiated Cells Challenges Its Role as a Pure Stem Cell Marker

The Oct‐4 transcription factor, a member of the POU family that is also known as Oct‐3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct‐4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct‐4 expression. On the contrary, we demonstrated, using reverse transcription‐polymerase chain reaction (PCR) (total RNA, Poly A+), real‐time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct‐4. These observations raise the question as to whether the role of Oct‐4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct‐4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic‐specific gene with a large number of pseudogenes that may contribute to false identification of Oct‐4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct‐4 in fully differentiated cells.

Bone regeneration: stem cell therapies and clinical studies in orthopaedics and traumatology.

•  Introduction •  Characterization of cells for bone regeneration in human beings •  State of the art in bone tissue engineering ‐  Biomaterials for scaffolding mesenchymal stem cells ‐  Future trends in bone tissue engineering •  Clinical targets for cell therapy in orthopaedics ‐  Current clinical problems and therapeutic approaches ‐  Potential clinical applications of cell based therapies for bone repair ‐  Data reported in clinical studies •  Selected paediatric bone disorders and cellular therapies ‐  Inborn errors of bone metabolism and cellular therapy ‐  Degenerative bone disorders in childhood •  Ethical aspects of EU clinical trials ‐  Ethics related to information and consent ‐  Approval of the studies ‐  Ethics related to privacy/data protection ‐  Ethics related to the risk‐benefit assessment ‐  Ethics related to protection of the health of persons involved in clinical trials ‐  Ethics related to transparency regarding research results •  Future directions and remarks

What is beyond a qRT‐PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes

In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation‐related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT‐PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.

Myocardial blood flow and infarct size after CD133+ cell injection in large myocardial infarction with good recanalization and poor reperfusion: Results from a randomized controlled trial

Objective Large acute ST-elevation myocardial infarction (STEMI) sometimes leaves extensive ischemic damage despite timely and successful primary angioplasty. This clinical picture of good recanalization with incomplete reperfusion represents a good model to assess the reparative potential of locally administered cell therapy. Thus, we conducted a randomized controlled trial aimed at evaluating the effect of intracoronary administration of CD133+ stem cells on myocardial blood flow and function in this setting. Methods Fifteen patients with large anterior STEMI, myocardial blush grade 0–1 and more than 50% ST-elevation recovery after optimal coronary recanalization (TIMI 3 flow) with stenting were randomly assigned to receive CD133+ cells from either bone marrow (group A) or peripheral blood (group B), or to stay on drug therapy alone (group C). The cells were intracoronary injected within 10–14 days of STEMI. Infarct-related myocardial blood flow (MBF) was evaluated by NH3 positron emission tomography 2–5 days before cell administration and after 1 year. Results MBF increased in the infarct area from 0.419 (0.390–0.623) to 0.544 (0.371–0.729) ml/min per g in group A, decreased from 0.547 (0.505–0.683) to 0.295 (0.237–0.472) ml/min per g in group B and only slightly changed from 0.554 (0.413–0.662) to 0.491 (0.453–0.717) ml/min per g in group C (A vs. C: P = 0.023; B vs. C: P = 0.066). Left ventricular volume tended to increase more in groups B and C than in group A, ejection fraction and wall motion score index remained stable in the three groups. Conclusion These findings support the hypothesis that intracoronary administration of bone marrow-derived, but not peripheral blood-derived CD133+ cells 10–14 days after STEMI may improve long-term perfusion.

The Role of PET with 13N-Ammonia and 18F-FDG in the Assessment of Myocardial Perfusion and Metabolism in Patients with Recent AMI and Intracoronary Stem Cell Injection

Over the last decade, the effects of stem cell therapy on cardiac repair after acute myocardial infarction (AMI) have been investigated with different imaging techniques. We evaluated a new imaging approach using 13N-ammonia and 18F-FDG PET for a combined analysis of cardiac perfusion, metabolism, and function in patients treated with intracoronary injection of endothelial progenitors or with conventional therapy for AMI. Methods: A total of 15 patients were randomly assigned to 3 groups based on different treatments (group A: bone marrow–derived stem cells; group B: peripheral blood–derived stem cells; group C: standard therapy alone). The number of scarred and viable segments, along with the infarct size and the extent of the viable area, were determined on a 9-segment 13N-ammonia/18F-FDG PET polar map. Myocardial blood flow (MBF) was calculated for each segment on the ammonia polar map, whereas a global evaluation of left ventricular function was obtained by estimating left ventricular ejection fraction (LVEF) and end-diastolic volume, both derived from electrocardiography-gated 18F-FDG images. Both intragroup and intergroup comparative analyses of the mean values of each parameter were performed at baseline and 3, 6, and 12 mo after AMI. During follow-up, major cardiac events were also registered. Results: A significant decrease (P < 0.05) in the number of scarred segments and infarct size was observed in group A, along with an increase in MBF (P < 0.05) and a mild improvement in cardiac function. Lack of infarct size shrinkage in group B was associated with a marked impairment of MBF (P = 0.01) and cardiac dysfunction. Ambiguous changes in infarct size, MBF, and LVEF were found in group C. No differences in number of viable segments or in extent of viable area were found among the groups. At clinical follow-up, no major cardiac events occurred in group A patients, whereas 2 patients of group B experienced in-stent occlusion and one patient of group C received a transplant for heart failure. Conclusion: Our data suggest that a single nuclear imaging technique accurately analyzes changes in myocardial perfusion and metabolism occurring after stem cell transplantation.

Adipogenic potential in human mesenchymal stem cells strictly depends on adult or foetal tissue harvest

Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Whartons jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes.

Airway Fistula Closure after Stem-Cell Infusion

Investigators observed the healing of a broncholpeural fistula soon after the injection of mesenchymal stem cells into the area surrounding the fistula.

Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages

BackgroundHuman mesenchymal stem cells (hMSC) have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion.ResultsWe used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS) to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC) produced under conditions of good manufacturing practices (GMP). Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z) 8950, 10087, 10345, and 13058) through subculture steps (P0-P7) with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin).ConclusionsThis study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion.

Endothelial Colony Forming Capacity is Related to C-Reactive Protein Levels in Healthy Subjects

The majority of clinical studies on endothelial progenitor cells (EPCs) focuses on the role of these cells in cardiovascular diseases and no systematic studies exist regarding their variations in healthy subjects. In order to define the burden of angiogenesis in physiological conditions we assessed the frequency of peripheral blood endothelial colonies (PB-ECs) and their relation with other factors possibly involved in their function such as high-sensitivity C-reactive protein (hs-CRP), endothelial cell-specific mitogen factor (VEGF) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in a highly selected healthy population. A PB sample was obtained from 37/47 healthy subjects (age 40.2+/-15.0yrs; M/F 15/22) without known cardiovascular risk factors. The serum level of hs-CRP, VEGF, TIMP-1, the frequency of PB-ECs by clonogenic assay, and the number of early EPCs and late EPCs by flow cytometry analysis were evaluated. PB-ECs were formed by 40.5% of studied subjects with a mean of 0.40+/-0.82 colonies/10(6) cells. The differences in the frequency of colony formation between genders were not statistically significant. The subjects with PB-ECs were characterized by higher values of hs-CRP, when compared with those not forming colonies, 0.276+/-0.230 vs 0.095+/-0.077 mg/l (p=0.003) respectively, and of VEGF, 328.3+/-162.9 vs 202.68+/-118.53 pg/ml (p=0.02). No significant differences were found in TIMP-1 values. The EPC clonogenic potential seems to be related to hs-CRP and VEGF levels even in healthy population supporting the concept that these mediators are involved in physiological ECs function.

Autologous mesenchymal stem cell therapy for progressive supranuclear palsy: translation into a phase I controlled, randomized clinical study

BackgroundProgressive Supranuclear Palsy (PSP) is a sporadic and progressive neurodegenerative disease which belongs to the family of tauopathies and involves both cortical and subcortical structures. No effective therapy is to date available.Methods/designAutologous bone marrow (BM) mesenchymal stem cells (MSC) from patients affected by different type of parkinsonisms have shown their ability to improve the dopaminergic function in preclinical and clinical models. It is also possible to isolate and expand MSC from the BM of PSP patients with the same proliferation rate and immuphenotypic profile as MSC from healthy donors. BM MSC can be efficiently delivered to the affected brain regions of PSP patients where they can exert their beneficial effects through different mechanisms including the secretion of neurotrophic factors.Here we propose a randomized, placebo-controlled, double-blind phase I clinical trial in patients affected by PSP with MSC delivered via intra-arterial injection.DiscussionTo our knowledge, this is the first clinical trial to be applied in a no-option parkinsonism that aims to test the safety and to exploit the properties of autologous mesenchymal stem cells in reducing disease progression. The study has been designed to test the safety of this “first-in-man” approach and to preliminarily explore its efficacy by excluding the placebo effect.Trial registrationNCT01824121