Mariacarla Iorio

Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells

Background -Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.Findings -MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.Conclusion -Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Isolation and characterization of equine amnion mesenchymal stem cells

The amnion is a particular tissue whose cells show features of multipotent stem cells proposed for use in cellular therapy and regenerative medicine. From equine amnion collected after the foal birth we have isolated MSCs (mesenchymal stem cells), namely EAMSCs (equine amnion mesenchymal stem cells), from the mesoblastic layer. The cells were grown in α-MEM (α-modified minimum essential medium) and the effect of EGF (epidermal growth factor) supplementation was evaluated. To assess the growth kinetic of EAMSCs we have taken into account some parameters [PD (population doubling), fold increase and DT (doubling time)]. The differentiation in chondrogenic, adipogenic and osteogenic types of cells and their epitope expression by a cytofluorimetric study have been reported. EGF supplementation of the culture medium resulted in a significant increase in PD growth parameter and in the formation of bone nodules for the osteogenic differentiation. By immunohistochemistry the amnion tissue shows a positivity for the c-Kit (cluster tyrosine-protein kinase), CD105 and Oct-4 (octamer-binding transcription factor 4) antigens that confirmed the presence of MSCs with embryonic phenotype.

Pre-birth selection of umbilical cord blood donors.

BACKGROUND . The fact that only a small percentage of cord blood units (CBU) stored are actually used for transplantation contributes to raising the already high costs of their processing and cryopreservation. The identification of predictors allowing the early identification of suitable CBU would allow a reduction of costs for the collection, storage and characterisation of CBU with insufficient volume or cell numbers. In our bank we have adopted a cut-off value for using CBU of 8 x 10(8) nucleated cells and a volume >or= 60 mL. MATERIALS AND METHODS In 365 banked CBU, we evaluated the correlation between neonatal/gestational parameters and laboratory data used to assess their quality. RESULTS Biparietal diameter (BPD) and abdominal circumference were significantly and positively correlated with CBU volume (r(2)=0.12, p=0.0011 and r(2)=0.092, p=0.0063, respectively). Receiver operating characteristic (ROC) analysis showed that both parameters can be used to identify CBU with insufficient volume (BPD: area under the curve 0.69, 95% CI=0.57-0.82, p=0.004; abdominal circumference: area under the curve 0.67, 95% CI=0.54-0.79, p<0.01). BPD and head circumference, but not abdominal circumference or femoral length, were positively correlated with white blood cell (WBC) count (r(2)=0.215, p=0.031, and r(2)=0.299, p=0.015, respectively). Abdominal circumference, but not BPD, head circumference or femoral length, was statistically significantly correlated with the number of CD34(+) cells in the CBU. Weight at birth and placental weight were positively correlated with WBC count, blood volume, CD34(+) cell count, total colony-forming units and burst-forming units. CONCLUSION . Pre-birth assessment of BPD might allow the selection of donors who would yield CBU of sufficient volume and WBC count and avoid the costs of collecting, transferring, storing and analysing CBU with a high probability of resulting unsuitable for transplantation.

Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood.

BACKGROUND In addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel. MATERIALS AND METHODS Cord blood units collected at public banks with total nucleated cell counts <1.5×10(9), platelet count >150×10(9)/L and volume >50 mL, underwent soft centrifugation within 48 hours of collection. Platelet-rich plasma was centrifuged at high speed to obtain a CBPC with target platelet concentration of 800-1,200×10(9)/L, which was cryopreserved, without cryoprotectant, below -40 °C. RESULTS During 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10(9)/L. Total platelet count per CBPC was 11.3±4.9×10(9). Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC. DISCUSSION This study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.

Knowledge about umbilical cord blood donation: a survey-based study performed in Tuscany.

Dear Sir, It is well known that umbilical cord blood (UCB) is an alternative source of stem cells for haematopoietic transplantation, which can cure children and adults of malignant and non-malignant diseases, such as leukaemias, lymphomas, metabolic disorders, immunodeficiency states, haemoglobinopathies and bone marrow failure syndromes1–2. Cord blood processing and cryopreservation are very costly procedures. In order to reduce these costs several pre-birth parameters were suggested to be useful for identifying and selecting the potential best collections3 . A survey-based study was recently performed in Greece on a sample of Greek citizens, with high reproductive capacity. The results indicated that the majority of them (74%) were in favour of UCB storage and that the main source of information (45.5%) was the media, with an additional source provided by medical health care professionals (36.6%)4. However, most of the Greek population do not have adequate information about this precious resource and the authors believed that different campaigns regarding UCB storage and uses should be developed through the cooperation of the Ministry of Health and Social Solidarity, the National Blood Donation Centre and the National Transplant Organisation4. In accordance with the findings of the Greek study, a survey conducted by Screnci et al. at the “Sapienza” University of Rome on blood donors and pregnant women showed that a considerable proportion of the individuals analysed had some general, but minimal knowledge about UCB. Moreover, the level of knowledge regarding the proper and real usefulness was generally low: 31% of informed pregnant women gave a wrong answer. A very small proportion of them consulted the specific website section of the Ministry of Health concerning the appropriate use of stem cells from UCB5. Despite the theoretically positive intention of Greek respondents towards UCB storage, most did not proceed to do so. Among the parents who chose to store UCB, most preferred private storage, perhaps because of the misleading information that exists in Greece about public banks in contrast to private banks. The reason given for choosing private banks was that the UCB would be stored better. Moreover, private storage was encouraged by doctors, friends or relatives and by the belief that it guarantees the baby’s future4. A survey-based study was conducted from the 1st September 2013 to the 31st of January 2014, in the whole region of Tuscany. The aim of the survey was to explore the knowledge that pregnant women have about UCB stem cell donation and the quality of education from health care professionals. For this purpose anonymous, standardised printed or on-line questionnaires were provided. Printed questionnaires were given to 2,999 women (average age=31.91) who delivered in birth centres in 14 Hospitals, while on-line questionnaires were sent to gynaecologists and 706 midwives who are involved in raising awareness about donation and cord blood collection. The surveyed population was divided based on the birth centre of origin (Table I). Table I Distribution of the sample. Since only eight gynaecologists filled in the questionnaire, our attention focused on pregnant women and midwives. Among 2,999 mothers who were asked to fill in the questionnaire, 1,568 (52%) returned the completed form; 60% of the respondents had not had previous pregnancies. The women were asked whether they had received general information about UCB donation during pregnancy. A considerable portion of pregnant women (82%) were aware of the possibility of donating UCB, while 18% had not received any information. The most common sources of information came from midwives (41.90%), gynaecologists (17.16%), family doctors (1.4%) and other sources, such as friends (14.60%) and media (7.53%). Several women received information from more than one of these sources. The 706 midwives involved in this study were divided in different groups depending on how long they had worked. While 70.49% of them had not received specific training on cord blood donation during their degree course, 69.4% of the midwives had attended a post-degree course about cord blood donation. Overall, 68.31% of midwives claimed to promote cord blood donation; however, although they claimed to be competent enough (mean=6.4) about their own skill and knowledge, 95.63% of them believed that further specific cord blood training would be necessary. About three-quarters (72.60%) of midwives use printed leaflets to give information, while only 1.37% of them use verbal communication. The majority of midwives spend an average of 13 minutes to give information. The critical aspects that arose from the survey highlight some solutions that could be adopted in order to improve the quality of information provided to pregnant women. The lack of interest shown by gynaecologists is one aspect which limits the choice of donating UCB. For this reason further involvement of these professionals will be necessary in the future. Midwives who had been employed for longer did not receive specific training on UCB collection during their degree course, even if they showed greater participation in training courses, in comparison with their colleagues who had started work more recently. However, all midwives declared that specific training courses would be important to update their knowledge on UCB donation and to make colleagues aware of this topic. Moreover, the time spent by midwives to inform pregnant women is thought to be insufficient. Finally, we consider that divulgation of information by means of leaflets should be combined with verbal communication, which is known to be the best way of raising people’s awareness. We believe that in our regional reality Hospitals should invest more money and time in promoting UCB donation, mostly because our data show that 58% of midwives don’t usually provide with information about this topic. Finally, we believe that professionals and institutions should make an effort to provide updates, education and evidence-based information about UCB donation, focusing on both the usefulness and limitations of UCB stem cells.