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In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.

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.

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.

Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated. For a possible clinical application, all experiments were carried out in serum-free media. A low-affinity nerve growth factor receptor (LNGFR) pseudotyped murine retroviral vector was used to transduce cord blood CD34+ cells, which were then expanded ex vivo. These cells engrafted up to three generations of serially transplanted nonobese diabetic/severe combined immunodeficiency mice: 54.26% ± 5.59%, 19.05% ± 2.01%, and 6.15% ± 5.16% CD45+ cells from primary, secondary, and tertiary recipient bone marrow, respectively, were LNGFR+. Repopulation in secondary and tertiary recipients indicates stability of transgene expression and long-term self-renewal potential of transduced HSCs, suggesting that retroviral gene transfer into HSCs, followed by ex vivo expansion, could facilitate long-term engraftment of genetically modified HSCs.

Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular homeostasis; unfortunately, the response of EPCs in physiological conditions remains largely unknown. Herein we report our observations of a 44-year-old healthy subject after a trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are strong stimuli for clonogenic endothelial cell activation and activity, as shown by the increase in the number of mature EPCs and in the endothelial colony-forming unit capacity. Both of these effects were completely reverted at sea level, 45 days after the subject's trek. Am. J. Hematol. 79:76-78, 2005. 2005 Wiley-Liss, In

Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cells for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both stem cell sources, we performed a comparative analysis of gene expression of CD133+ cells from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray results are strictly dependent on the purity of the cell population and on the RNA quality, rigorous quality controls were applied. The purity of CD133+ cells was more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cut-off for significance has been determined tuning the Delta parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. This finding could justify the peculiar immunological properties of CB. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, maybe due to the neonatal origin of these cells. In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more susceptible to different stimuli and potential fates.

The selective homing of peripherally injected marrow MNCs (MMNCs) has recently been demonstrated in a model of cryodamaged heart. However, the mechanism underlying this phenomenon is still unknown. In the hypothesis that this process is related to the necrotic area extension, the infarcted area was correlated with the number of homed MMNCs in a model of experimental cryodamaged heart.A total of 12 donor and 12 recipient inbred isogenic adult (4 weeks old) Fisher rats were used to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by cryoinjury. MMNCs were purified, labeled with PKH26 (a red fluorescent cell dye), and infused 7 days after the injury through the femoral vein of recipient rats. One week after peripheral administration, the number of homed MMNCs was assessed and the infarct size was correlated with the number of cells present in the target.Labeled cells were found only in the injured myocardium of the treated animals (n = 6), where a mean of 12 +/- 3 PKH26+ cells per section examined were found; a significant correlation was found between the infarct size and the estimated number of cells (p = 0.008)These data indicate that the homing of MMNCs is related to the extent of the myocardial injury, suggesting that cellular therapy for regeneration of damaged myocardium should be individualized by taking into consideration the extension of the area to repair.

Significant progress has been achieved during the past 10 years in cell transplantation and recent research has focused on the possibility of improving ventricular function after myocardial infarction. Most studies in the field of cardiac tissue repair are performed by direct intramyocardial injection of cells of different origin. Since this approach requires a surgical intervention, in this study we investigated the feasibility of non-invasive administration of bone marrow mononuclear cells (BMMNCs) by assessing the fate of peripherally injected, purified, labeled cells in cryodamaged hearts.Ten donor and ten recipient inbred isogenic adult (4 weeks old) Fisher rats were used as models to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by placing a frozen metal probe on the anterior left ventricular wall for 15 seconds (freeze-thaw injury technique). BMMNCs were purified and labeled with a red fluorescent cell dye. Seven days after the injury about 15-25x10(6) cells were infused through the femoral vein of recipient rats. Seven days after the infusion, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells.Labeled cells were found only in the injured area of the heart and not in the normal tissue, and a limited number of cells were identified in the spleen of all the animals. Most of the labeled cells in the infarcted area were Thy-1(+) and some were CD34(+).Our data suggest that peripherally injected BMMNCs can traffic through the circulation to the site of damage; we hypothesize that tissue injury leads to the priming of a cytokine cascade acting as chemoattractant for the infused cells.

Background: Progressive 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/design: Autologous 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. Discussion: To 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 registration: NCT01824121

We describe the activities of the Milan Cord Blood (CB) Bank and of the Italian Cord Blood Network. By October 31, 1995, 763 units were banked in Milan. Of these, 8 units were used to perform 4 related and 4 unrelated transplants in 5 children and 3 adult patients. Early cord clamping after delivery was found to be crucial to increase the volume of CB collected. This procedure does not seem to be detrimental to the newborn. Of various red cell sedimenting agents used to reduce the unit volume and concentrate progenitor cells, 3% gelatin seems to be associated with the best yields. After a preliminary experience of 2 years, the Italian Cord Blood Network (Gruppo Italiano Amplificazione Cellule Emopoietiche, GRACE) was founded in 1995. The initial activities of GRACE are aimed at the development at the national level of CB banking standard operative procedures in agreement with the draft issued by The North American Task Force for The Development of Standards for Hematopoietic Cell Transplantation. Moreover, a wet workshop has been organized to standardize colony-forming unit (CFU) evaluation. The main goal in Milan is the collection of 5000 CB units. Other issues of interest include CB volume reduction, hematopoietic progenitor purification, ex vivo expansion prior to transplantation, and experimental protocols for gene transfer, such as those related to the multidrug resistance (MDR) gene.

Autologous- or allogeneic-bone marrow transplantation are increasingly used to overcome the myelosuppressive effects of high dose chemotherapy administered to cancer patients. Transfer of the multidrug resistance (MDR) gene in hemopoietic progenitors has been proposed as a tool to administer higher and possibly more curative doses of chemotherapy. Murine models have demonstrated that retrovirus-mediated MDR transfer in bone marrow cells can render animals resistant to myeloablative doses of Taxol, and in vitro studies have shown that MDR-transduced human CD34+ cells can generate drug-resistant multipotential hemopoietic progenitors such as long term culture-initiating cells. Given these results, phase I clinical trials are currently under way to evaluate feasibility and treatment-related toxicity of MDR gene transfer in cancer patients by means of safe retroviral vectors. Finally, Taxol treatment of MDR transduced mice and human CD34+ cells have indicated that MDR is a dominant selectable marker in vitro and in vivo, and vectors carrying both MDR and non selectable genes such as beta-globin or glucocerebrosidase could be used in the next future for gene therapy of inherited disorders like thalassemia or Gaucher disease.

Clinical-grade cell purification from thawed cord blood: an example of translational research

Summary. We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer, Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, effciency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 μg/ml taxol. In uninfected control, 1–2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14–31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometiric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotien, was found on 17–25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Abstract 4403 Our hospital-based GMP facility, named Cell Factory, built in Milano (Italy) and the first one authorized by the National Drug Agency (AIFA) in 2007, has pioneered innovative manufacturing technologies in order to supply therapeutic clinical development programs. By our group and by others, cord blood (CB) mesenchymal stem cells (MSC) have been already tested both in vitro and in extensive pre-clinical disease models, demonstrating that CBMSC are safe and effective. Therefore, CBMSC are the best candidate product for several clinical trials including neurologic and renal repair (1–3). In this context, our group developed the first off-the-shelf (OTS) bank where CBMSC are expanded and banked in GMP conditions, and now they are available as “a drug”, to be distributed for patient doses, much like an off-the-shelf pharmaceutical product. From informed consent CB donations, CBMSC were grown and expanded in CellSTACK kit that includes devices for a closed-system cell culture (Macopharma) with EMEA-grade Australian FBS and they were manufactured through a multi-step controlled and well-validated process. Cell Factory established this innovative culture approach through sufficient passages to produce hundreds of thousands of potential doses from an individual donor. After culture, our quality control approach has been designed to ensure that controls are implemented and completed satisfactorily during manufacturing operations, and that developed procedures and specifications are appropriate and followed, including those performed by contract testing laboratories. In fact, at each step and at the end of the production process before cryopreservation, CBMSC were analyzed and qualified according to pre-established criteria (cell count, viability, extensive immunophenotype, CFU-F, karyotype, CGHArray, telomerase activity, sterility, mycoplasma and endotoxin assays) to ensure that a consistent, well characterized product candidate is produced. The same CBMSC batches can be provided for pre-clinical testing and for further validations to prepare the investigational medicinal product dossier (IMPD) for each clinical trial approvals. Now these GMP batches of CBMSC are cryopreserved and available in our OTS CBMSC bank in different cell doses to comply any kind of medical needs. Products are provided under specific contract conditions developed with the clinical centers. (Contact: cellfactorypoliclinico@gmail.com) Disclosures: No relevant conflicts of interest to declare.

Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p Disclosures: No relevant conflicts of interest to declare.

Umbilical cord blood has been recently used as a source of hematopoietic progenitor cells for transplantation of pediatric patients. This study was performed to evaluate the feasibility of a cord blood bank for unrelated transplant. When the umbilical cord was clamped within 20 seconds after delivery, it was possible to collect 86±25 ml of cord recipients with more than 2000 CFU-GM/kg; 53% of cord blood samples were found to contain enough CFU-GM for engraftment in 50-70 kg adult patients.

Abstract 4446 Hematopoietic stem and progenitor cells are routinely used in onco-hematological applications as consolidated approaches. Recently, these cells have been exploited also for their non-hematopoietic potential in the novel field of regenerative medicine. In fact, several clinical trials are already ongoing in the cardiovascular field, and more and more innovative applications, such as in liver disorders, are emerging to treat patients for whom no other efficient therapies are available. In Europe, the preparation of hematopoietic cells for non-hematopoietic applications must comply to medicinal products current good manufacturing practices (cGMP) and thus, the production process and the analytical methods applied in quality controls must be validated in order to demonstrate standardization and compliance with pre-defined specifications. In our hospital-based GMP facility, named Cell Factory, the first one authorized by the National Drug Agency, we have several processes ongoing for different clinical experimental trials. In particular, we produce CD133+ cells from leukapheresis (AF) products for liver chronic disease and bone marrow (BM) CD133+ cells for cardiovascular repair, strictly following the GMP rules governing medicinal product and we validated both processes as herein reported. In our hands, AF and BM need different approaches to improve the purity of the final cell product that is crucially important to have consistent clinical results. In fact, BM requires an efficient erythrocyte depletion that we obtained by optimizing a centrifugation based separation system, before the CliniMacs (Miltenyi, Gladbach, Germany) immunomagnetic purification. To validate the aseptic technique, three runs of specific media-fill protocols were set up. Finally, to validate the defined protocol, three runs of validation were performed in our class B facility for both procedures with all the quality controls on “in process” as well as on final products. BM samples from 3 patients who underwent sternotomy for by-pass surgery were collected after informed consent and processed as previously described. The results of the three validation runs were consistent in: cell dose >2×106 (2.54×106, 2.81×106 and 2.62×106), purity >70% (87.5%, 97.4% and 93.6%), viability >70% (88.5%, 90.6% and 96.4%), no growth for sterility and endotoxin AF samples from 3 patients who underwent mobilized standard-volume leukapheresis were collected and processed. The results of validation processes were consistent in: cell dose >100×106 (181×106, 138.5×106 and 178.3×106), purity >80% (91%, 90% and 92%), viability >80% (89%, 93% and 95%), for sterility no growth and for endotoxin In conclusion, all the end-points were reached and currently the cell products are provided to different clinical sites as investigational new drug, approved by the national competent authority. These products, as well as other cellular therapy products, are available upon request at our facility for experimental clinical applications (www.cellfactorymilano.com). Disclosures: No relevant conflicts of interest to declare.

Abstract 2588 Mesenchymal stem cells (MC) represent a promising population for regenerative medicine. Recently, their presence has been documented not only in bone marrow, but also in cord blood (CB), an attractive stem cell source with no ethical controversy. In this study, we investigated if CB could represent a new therapeutic tool for the treatment of severe vascular diseases. We first validated a negative immunoselection approach using a cocktail of antibodies that targets and reduces erythroid components in order to efficiently isolate low-potential (lp)-CBMSC (46% of isolation efficiency) and high-potential (hp)-CBMSC (8.1% of isolation efficiency). Specific aim of this study was to investigate the proangiogenic effect of hp-CBMSC for endothelial regenerative purposes. Previous experiments had shown that lp-CBMSC are able to grow until passage 4–5 only, with a lower differentiation rate as compared to the hp-CBMSC, therefore we used hp-CBMS. First, they were induced towards the endothelial lineage by growing them in endothelial medium supplemented with 5% FBS, 100 ng/ml Vascular Endothelial Growth Factor (VEGF) and 50 ng/ml Endothelial Growth Factor (EGF) in 24-well cell culture plates coated with fibronectin. After 3 weeks of induction, the ability of induced-cells to self-organize into capillary-like structures in the presence of basement membrane matrix was analyzed. Human umbilical vein endothelial cells (labeled with green fluorescent dye PKH67) were co-cultured with induced (A) or non-induced (B) hp-CBMSC (labeled with red fluorescent dye PKH26) in the presence of hp-CBMSC conditioned media (supernatant collected from hp-CBMSC cell culture). Interestingly, the tube formation was observed only in the presence of induced-hp-CBMSC (A) where the contribution of the endothelial (green) and mesenchymal (red) compartment in the vascular network was extremely clear and evident. In addition, to evaluate whether there was a possible endothelial contribution of the conditioned medium released by the non-induced-hp-CBMSC, an angiokit assay (Angiokit, TCS CellWorks Ltd) was performed. A strong tube formation was observed and the anastomosis-like structure was confirmed by the expression of CD31 and Von Willebrand Factor (DAB staining) in immunocytochemistry. The next step was to identify the factors released in the conditioned medium and in fact several angiogenic factors were found: HB-EGF 13.5 (8.7 – 16.7); HGF 22.4 (16.3 – 39.1); FGF 10.6 (7.0 – 13.5); VEGF 471.3 (369.3 – 590.0) (the results are expressed as median (and range) pg/mL and normalized per 1×106 seeded cells) using a cytokine array (Searchlight Multiplex Immunoassay Kits). The work is still ongoing for the quantification of the endothelial gene expression by real-time PCR for some angiogenic genes including: angiopoietin-2, the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular endothelium-selectin (E-selectin), VCAM1 and VEGF-A in the induced versus the non-induced hp-CBMSC. In conclusion, our promising results suggest that hp-CBMSC can be considered as endothelial “drug stores” and are extremely promising paracrine effectors that could be exploited in future clinical applications for endothelial tissue regeneration. Disclosures: No relevant conflicts of interest to declare.

To the Editor: In a recent issue of Blood, Guenechea et al[1][1]reported that ex vivo cytokine-expanded CD34+ cord blood cells retained nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating activity of fresh cells, although there was a significant delay in the time to achieve

Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

Bronchopulmonary dysplasia is a chronic lung disease that occurs in very premature infants and is characterized by impaired alveologenesis. Despite the use of surfactant, antenatal glucocorticoid treatments and new ventilation strategies, mortality in these subjects is still high and the impairment of alveolar formation in the surviving premature infants leads to abnormal lung function that induces neurological and growth dysfunction. In this context innovative cell-based lung repair approaches are highly needed. In particular, it would be important to develop a system that could regenerate the alveolar type I cells (ATI), that cover about 95% of the alveolar surface, and the alveolar type II cells (ATII). ATII cells ensure surfactant production, which decreases the surface tension of the alveolar surface, allowing the alveoli to expand during inspiration and preventing the collapse during expiration. Moreover, a sufficient ATII cell number is important because they serve as stem cells for ATI cells. A very promising, still poorly known and easily available, stem cell source is the human umbilical cord (HUC). We isolated and characterized a cell population within the HUC of pre-term newborns (HUC perivascular cells) to evaluate their capability to induce or improve pneumocyte repair after damage. With this aim we processed the HUCs (n=14) from deliveries of consenting mothers (23–32weeks of gestation). Pieces of cord were dissected to expose the Wharton’s Jelly and the vein and the two arteries were exposed. The ends of each dissected vessel were tied together and digested with Collagenase A for 18 hours. After digestion the cells were extensively characterized by flow cytometry. The median (and range) percentage of the cells with a phenotype consistent with perivascular/pericyte-like cells (CD146+/CD45−/CD34−) was 17.5 (7.6–39). Moreover, these cells were positive for alpha-SMA (median and range: 90.7%, 46.9–94), NG2 (median and range: 23.2%, 13.05–31.8), VeCad (median and range:10.8%, 2.6–32.4), KDR (median and range: 15.25%, 1.5–26.2), CD31 (median and range: 3%, 0.8–8.7) and negative for CD45 and CD105. The HUC perivascular cells were cultured in the presence of EGM2 on a pre-coated gelatin layer for 1 week, and then with DMEM +20%FBS and maintained in long-term culture (11 passages). The cells were then stained with the fluorescent dye PKH26 and co-cultured in transwell with the ATII epithelial rat cellular line damaged by bleomycin. Control cultures were set up with not-damaged cell line. After 4-day co-culture, as a result of the chemiotactic migration, we observed PKH26 positive cells in contact with the damaged alveolar cell layer and not in the control cultures. By immunocytochemistry these cells showed to co-express PKH26 and the typical markers (pro-surfactant protein C, cytokeratin 18) of the ATII cells. Our results suggest that it is possible to isolate and to maintain in long-term culture HUC perivascular cells from pre-term cords. Moreover, this population is able to migrate towards alveolar cell layer previously damaged with bleomycin and to express the markers of the ATII cells. Our data encourage further investigations in order to evaluate the feasibility of therapeutic applications of these cells.

Introduction: Human cord blood (CB) represents an interesting source of mesenchymal stem cells (MSCs) with different cell “competence” and feasible clinical applications. Up to now patients affected by acute renal failure (ARF) are treated by unsuccessful pharmacological therapies and it is an interesting possibility that CB MSCs or other potential progenitor cell subpopulations could be used for therapeutic purpose in renal repair. Aim of this study was to isolate and culture MSCs from full-term UCB and to test their ability to promote renal repair when transplanted into NOD-SCID mice with acute renal failure. Methods: MSCs were isolated with a lineage-depletion negative immuno-selection procedure (Rosette Sep, StemCell Technologies, Vancouver, Canada) from buffy coat of CB (n=17) without lysis of red blood cells. Cell suspension was seeded at 1×106 cells/cm2, in 35mm/tissue culture dish in the presence of αMEM, 20 % FBS, 2mM L-Glutamine and Penicillin/Streptomycin. Immunophenotype was evaluated by flow cytometric analysis while the differentiation assays were performed towards adipogenic, osteogenic and chondrogenic lineages in order to confirm their mesenchymal features. To induce acute renal failure, NOD-SCID mice (n=10) were injected with cisplatin (12.7 mg/KG s.c.). This drug is associated with renal function deterioration, measured as serum blood urea nitrogen (BUN), peaking at day 4–5. After one day the mice received i.v. saline or 5×105 CB MSCs. Renal function and histology were evaluated. Results: The success rate of isolating MSCs from CB units was 17.6%. Flow cytometric analysis showed that MSCs were positive for CD44 (69%), CD105 (26%), CD90 (99%), HLA class I (80%) and negative for CD31, CD45, CD34, HLA class II. Moreover, within the CB MSCs we identified a subpopulation (37.5%) characterized by CD146+/34−/45− and consistent with perivascular/pericyte-like cells. Moreover we demonstrated and confirmed that CB MSCs were capable to differentiate in osteogenic and condrogenic but not adipogenic lineages, as recently shown also by other groups. In vitro differentiation towards epithelial lineage is in progress. In vivo results showed that CB MSCs significantly protected cisplatin-treated mice from renal function impairment at day 4 (BUN: cisplatin+saline 115±5 vs cisplatin+CB MSCs 64±13 mg/dl, p Conclusion: These preliminary results indicate that human CB MSCs exhibit reparative potential in acute renal failure. More evidences demonstrating the plasticity of CB stem cells will provide an exciting opportunity to explore the use of this stem cell source in regenerative medicine for patients with renal disease.

The main limitations of CB transplantation (CBT) are represented, in comparison to bone marrow transplantation, by a lower probability of engraftment and a slower hematopoietic recovery which can favor the occurrence of fatal complications. Several studies have clearly documented that the higher the number of cells infused, the faster the rate of engraftment and the lower the risk of transplant-related mortality. In order to increase the number of CB cells infused, approaches based on ex vivo expansion of CB progenitor cells are, in principle, particularly promising. In this regard, we developed a clinical protocol to expand the number of CB hematopoietic progenitor cells (HPCs), based on the use of a cytokine cocktail including interleukin-6 (10 ng/ml), stem cell factor (50 ng/ml), Flt-3 ligand (50 ng/ml) and thrombopoietin (10 ng/ml), in the presence of a serum-free medium and 10% AB allogeneic plasma. This combination induced significant and sustained ex vivo expansion of primitive CB HPCs. After clinical grade immunoselection (CliniMACS, Miltenyi Biotech), CD34+ cells were cultured at a concentration of 5x103 cells/ml for 2 weeks. Cytokines were added at the onset of culture and replaced twice a week. The purity of CD34+ cells (median and range) after immunoselection of thawed CB samples was 79.5% (47–96). After culture, the median fold expansion of total nucleated cells (NC) and CFU-GM was around 2 logs, while that of CD34+ cells was around 1 log. The expanded cells had levels of endotoxin below the required limit of 0.05IU/mL (E.Ph.) and were negative for mycoplasma, bacterial and fungal contamination. Two children, one with thalassemia and one with sickle cell disease (SCD), were given ex vivo expanded CD34+ cells from an HLA-identical sibling. In both cases the conditioning regimen included busulfan, thiotepa and fludarabine, while GVHD prophylaxis consisted of cyclosporine-A. The first patient, a 6-year-old boy with thalassemia, was given 2 CB units derived from 2 HLA-identical fraternal (i.e. non syngeneic) twins (1 unmanipulated CB unit and 1 CB unit infused after ex vivo expansion). The fold expansions of NC and CD34+ cells were 623 and 56.6 respectively. The patient had neutrophil recovery (i.e. above 500/cmm) on day +16, while the platelet recovery (i.e. above 30,000/cmm) occurred on day +31. The second patient, a 7-year-old girl with SCD, was given 2/3 of the CB unit unmanipulated and 1/3 after ex vivo expansion. The fold expansions of NC and CD34+ cells were 189.3 and 30.5 respectively. Neutrophil and platelet recovery occurred on day +18 and +33 respectively. Neither acute nor chronic GVHD occurred in both patients, who are alive and disease free at 12 and 5 months after CBT respectively. In the first patient, chimerism evaluation showed the presence of erythroid and myeloid colonies, as well as of T cells, derived from the ex vivo expanded cells up to 7 months after CBT. Mesenchymal stem cells remained of patient origin in both cases. These preliminary results indicate that our expansion procedure is safe and able to ex vivo expand CB HPCs; the expanded cells seem to accelerate hematopoietic recovery and, for the first time, we demonstrate that these cells contribute to sustain hematopoietic recovery after CBT. Altogether these results could widen the applicability of CBT in adults.

There are experimental evidences that Endothelial Progenitor Cells (EPCs) is a pool of cells that could proliferate and differentiate into mature endothelial cells and that may contribute to endothelial repair, but no systematic studies exist regarding physiologic variations in EP circulating pool and in CFU-Endo capacity in healthy subjects. In this regard, since the level of EPCs may be a biologic marker for vascular function, we decided to characterize primitive and circulating EPCs and their relation with gender, aging and the capacity to form CFU-Endo in a selected healthy population. Each subject underwent a screening evaluation to exclude any underlying cardiac diseases. A 20mL sample of peripheral blood was obtained (h 11:00–12:00 AM) from 15 male and 17 female age matched subjects (mean age 41.6±15.9yrs). EPCs were assessed by flow cytometric analysis on 100,000 events per sample (FACScan, Becton Dickinson). Primitive EPCs, defined as CD45−/34+/133+ and CD45−/KDR+/144−, were 8±1.8SE/uL. Circulating EPCs, defined as CD45−/146+/31+/FCSlow and CD45−/146+/144+, were 18.72±2.5SE/uL. No correlation was found between these populations and aging, gender and BMI. Instead a significant decrease in CD45−/34+/133+ (2.9±0.6SE/uL) subpopulation was observed with aging (r = 0.45; p = 0.009). CFU-Endo were performed as described by Hill JM et al. (NEJM 2003) and scored by microscopy in blind by two operators. The confirmation of endothelial-cell lineage was performed by immunostaining for endothelial-specific antibodies (CD31+, CD105+, CD144+, CD146low, CD133−, vWf-). CFU-Endo were expressed as the mean number of colonies per well in 8–25 separate determinations for each subjects and only 15 subjects gave colonies (mean 2.1±0.05SE). CFU-Endo do not correlate with aging and gender in healthy subjects confirming that endothelial injuries and cardiovascular risk factors are a crucial endothelium stimulus. In fact, recently it has been reported that higher colony counts were found in subjects with various degrees of cardiovascular risks. In conclusion CD45-/34+/133+ cell population correlates with aging but since this population represents Hemato-Endo Precursors that include a majority of endothelial progenitors and a small number of hematopoietic stem cells, further studies will be needed to identify a subpopulation strongly predictive of CFU-Endo capacity and to assess its endothelial lineage switch.

Stable oncoretroviral gene transfer into hematopoietic stem cells (HSC) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSC to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells the role of ex-vivo expansion was investigated. For a possible clinical application all experiments were done according to good anufacturing practice (GMP) guidelines. The combination of Flt3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO), and Interleukin-6 (IL6) has been shown to stimulate proliferation and self-renewal of very primitive (SCID-repopulating cells, SRC) hematopoietic cells. We asked whether it is possible to efficiently transduce HSC with oncoretroviral vector, to expand them and whether transduced cells retain their self-renewal potential, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice A Gibbon ape leukemia virus (GALV)-pseudotyped vectors already approved for clinical application has been used to efficiently and durably deliver a defective, non functional form of the cell surface marker truncated low affinity nerve growth factor receptor (LNGFR) into primitive cord blood (CB) HSC. The transduction was performed, following an up-to 24 hour exposure to FL, TPO, IL-6 and SCF, in the presence of the growth factors in serum-free (SF) medium on retronectin (RT) coated plates. At day 3 post-transduction, total cells and CD34+ cells were expanded 24-fold and 8.5-fold respectively. More than 40% of the cells were CD34+. Transduction efficiency was >55%. Serial transplantation is the most reliable method to assess the stable expression of a gene in cells with high proliferative potential. Mice transplanted with transduced or mock-transduced, expanded cells showed higher levels of human engraftment than those transplanted with unmanipulated cells (56.7%, 55% and 39.9% respectively). LNGFR expression of CD45+ cells was 14.35± 4.27%. All secondary mice transplanted with cells from primary recipient BM resulted engrafted (21.7%, 13.5 and 2.8% respectively). LNGFR expression was 47.54± 3.1% respectively of human CD45+ cells. BM cells from secondary recipients were used for tertiary transplants. Only mice transplanted with expanded cells were positively engrafted. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and expanded cells, showed good levels of human engraftment (6.15% CD45+). LNGFR expression was 49.1± 4.4% of human CD45+ cells. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34+), B (CD19+), myeloid (CD14+), erythroid (GpA+) and megakaryocyte cells (CD41+) in equivalent proportion. BM of the engrafted mice was placed in a human colony assay. Human colonies also were generated from the murine BM. In conclusion, we have validated a SF-protocol for efficient gene transfer into human CB HSC using a retroviral vector. Under these conditions, transduced and expanded cells repopulated NOD/SCID mice for 3 generations with a human multilineage graft stably expressing the transgene. In a view of future clinical applications, this protocol represents a major step towards the achievement of this goal.

CD34+ cells purified from 2 cord blood (CB) units using immunomagnetic columns were cultured in stroma free liquid culture at a concentration of 5×10 3 cells/ml in flask (10 ml/flask) for 2 weeks at 37°C in 5% CO 2 in the presence of interleukin (IL) -6 (10ng/ml), IL-11 (10ng/ml), Flt-3 ligand (50ng/ml) and thrombopoietin (10ng/ml). We investigated the immunophenotype of ex-vivo expanded CB− CD34+ cells at the onset of culture (T0) and 2 weeks later (2w) using three-colours flow cytometry and the following MoAbs: anti -CD34, -CD38, -CD33, -CDw90 and CD13 (Beckton-Dickinson). The table reports the percentages of CD34+ cell subsets: The expansions of CB-units 1 and 2 were: nucleated cells 2.63 and 2.4 log; CD34+ cells 1.78 and 1.76 log. These culture conditions determined a marked myelo-monocytic lineage commitment. However, CD38 was not expressed on 8.7 and 72% of CD34+ cells at 2w. These data support that CD8 may not be the hallmark of uncommitment during CD stem cell ex vivo expansion.

Clinical use of ex vivo expanded hematopoietic stem cells (HSCs) from cord blood (CB) carries important regulatory implications. In previous studies we identified a cytokine cocktail, (interleukin (IL)-6 (10 ng/ml), IL-1 (10 ng/ml), Flt-3 ligand-FL-(50 ng/ml) and thrombopoietin—TPO-(10 ng/ml) suitable to induce significant and sustained ex vivo expansion of CD34+/38− cells from CB for up to 10 weeks. In this study we expanded 4 samples each with 3 commercial media (A: StemPro 34 SFM, Life Technologies; B: CellGro SCGM, CellGenix; C: X-Vivo 10, BioWhittaker) in the presence of the same cytokine cocktail. CD34+ cells were purified from CB using an immunomagnetic column and cultured in stroma-free liquid cultures at a concentration of 5×10 3 cells/ml in 24-well plates for 2 weeks at 37°C in 5% CO 2 . The median and range fold expansions and the cell viability with different serum-free media are shown in the table: Based on these preliminary CD34+/38−/33− data, we elected to continue our studies with A and B media.

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.

Advanced therapy medicinal products represent a new generation of medicinal products for regenerative medicine. Since the implementation of the EU regulation for this innovative class of drugs, the academic and hospital institutions have played a central role in their development and manufacture. For these institutions that are not familiar with the industrial context, being in compliance with the pharmaceutical standards is extremely challenging. This report describes how we dealt with some specific issues during our hospital-based GMP experience. Furthermore, we identify as a future perspective the consistent stimulating contribution that a public entity can ensure for advanced therapy medicinal product development and licensing.

In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1β and TNFα synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.