The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPCs) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. To this end, hMPCs were isolated from a human muscle biopsy, and manipulated to ectopically express brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1). These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 106 hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury.

Introduction: Mesenchymal cells derived from human tissues are known for their anti-inflammatory characteristics, neuroprotective effect and modulation of the immune system. Glatiramer acetate (GA) long-acting formulation (GA Depot) consists of extended-release microspheres containing GA (the active ingredient in Copaxone©, indicated for RRMS treatment) for intramuscular (IM) injection. In this study we assessed the cell dose effect of ADSC injected intracerebroventricular (ICV) in a MOG-induced chronic EAE mouse model alone or with GA Depot, injected IM, on disease symptoms.

The copper chelator tetraethylenepentamine (TEPA; StemEx) was shown to attenuate the differentiation of ex vivo cultured hematopoietic cells resulting in preferential expansion of early progenitors. A phase I/II trial was performed to test the feasibility and safety of transplantation of CD133+ cord blood (CB) hematopoietic progenitors cultured in media containing stem cell factor, FLT-3 ligand, interleukin-6, thrombopoietin and TEPA. Ten patients with advanced hematological malignancies were transplanted with a CB unit originally frozen in two fractions. The smaller fraction was cultured ex vivo for 21 days and transplanted 24 h after infusion of the larger unmanipulated fraction. All but two units contained <2 × 107 total nucleated cells (TNCs) per kilogram pre-expansion. All donor–recipient pairs were mismatched for one or two HLA loci. Nine patients were beyond first remission; median age and weight were 21 years and 68.5 kg. The average TNCs fold expansion was 219 (range, 2–620). Mean increase of CD34+ cell count was 6 (over the CD34+ cell content in the entire unit). Despite the low TNCs per kilogram infused (median=1.8 × 107/kg), nine patients engrafted. Median time to neutrophil and platelet engraftment was 30 (range, 16–46) and 48 (range, 35–105) days. There were no cases of grades 3–4 acute graft-versus-host disease (GVHD) and 100-day survival was 90%. This strategy is feasible.

To examine whether ex-vivo expanded human bone marrow (BM)-derived AC133+ cells may participate in post myocardial infarction (MI) healing, we have examined the effect of the copper chelator tetraethylenepentamine (TEPA) on the ex-vivo expansion potential of BM-derived stem cell populations and the effect of the resulting ex-vivo expanded AC133+ cells in a MI animal model. AC133+ cells isolated from human BM, using the CliniMACS device, at purities greater than 90% were expanded in Teflon bags, in the presence of IL-6, TPO, Flt-3 ligand, and SCF with or without TEPA for three weeks. The progenitor cell composition and potential were examined at the end of the treatment time and after long-term incubation in culture. After 3 weeks in TEPA-treated cultures the total nuclear cell expanded more than 200±20 fold. The increase in the CD34+, AC133+ and AC133+/CD38-cell populations was 17±13, 16±1 and 270±110 - fold, respectively, and the CFU content was 84±28 fold higher than at the initiation of the cultures. Contrary to TEPA treated cultures, the cultures treated in the absence of the chelator lost their progenitor populations by week 5-7 in culture. Ex-vivo expanded AC133+ cells for 3 weeks expressed VEGF and VEGF receptor RNA as examined by RT-PCR.

In vitro cell expansion is constrained by default pathways of commitment and differentiation resulting in limited expansion of hematopoietic stem-progenitor cells (HSPCs). Still, several ex vivo manipulations have been reported to achieve expansion of HSPCs by altering cell cycle kinetics and enhancing progression through the G1-S barrier.

Ex-vivo expansion strategies of cord blood (CB) derived human progenitor cells (HPC) have been developed to provide an answer to the delayed time to engraftment and to the extended periods of neutropenia and thrombocytopenia encountered. These problems occur in transplants of CB products performed in adults due to the low yield of HPC. Reports correlating the clinical outcome with the number of CD34+ cells suggest that the transplantation of ex vivo expanded CD34+ cells may shorten the time to engraftment. The use of copper chelators such as tetraethylenepentamine (TEPA) has been shown to prolong expansion of HPC by inhibiting cell differentiation and thus allowing self-renewal of primitive HPC (Exp Hematol. 2004; 32:547). The variability observed in the expansion results, caused by the intrinsic differences among the various sources of CB units and processing methodologies, complicates the interpretation of published results. In the present report we summarize our results of CD34+ cell ex-vivo expansion of over 100 units in the presence of IL-6, TPO, Flt-3 ligand and SCF with and without TEPA. After 3 weeks, the total nuclear cell (TNC), colony forming unit (CFU), and the total CD34+ cell fold expansion of TEPA-treated cultures were 424±10.5 (n=230), 104±7 (n=112) and 19±3.2 (n=113), respectively, with no significant differences compared to controls. However, the percentage of the primitive subset of HPC, CD34+/38− cells, significantly (p<0.0001) increased in the TEPA-treated cultures (3.2%±0.2, n=59) vs. controls (1.6%±0.27, n=147). In contrast, after 5 weeks in culture, the TNC fold expansion was significantly (p<0.05) higher in TEPA-treated cultures compared to the controls, 1471±63.5 (n=89) vs. 1270 ±240 (n=55), respectively. The increase in TNC in TEPA-treated cultures did not result in increased HPC differentiation, but was accompanied by an increased self-renew capacity of CD34+ cells as represented by a 57±5.9 fold (n=47) vs. a 32±3.5-fold (n=38) amplification in the controls (p<0.0009). The overall fold expansion in culture analyzed by a Kaplan-Meier survival curve function demonstrate that the TNC, CFU, CD34+ and CD34+/38− cells derived from TEPA-treated cultures have higher in vitro survival probabilities than controls (p<0.0014). Cumulative values of all parameters were calculated and a transformation performed using the rank procedure. The results underline that TEPA increases CFU potential and CD34+ and CD34+/38− content during the ex-vivo expansion (p<0.01). The TEPA supplemented expansion technology was further tested after up-scaling of the processing and culturing procedures. AC133+ cells isolated by the CliniMACS device from frozen CB units obtained from 6 different banks. The expansion results of TNC, CD34+ cells, CFU and %CD34/38- were 337±23 (n=57), 21±4.3 (n=19), 133±27.5 (n=19) and 2.7% ±0.7 (n=19) fold, respectively. A clinical trial with TEPA expanded cultures for treatment of leukemia patients is currently ongoing at MD Anderson Cancer Center, USA.

Cord blood (CB) is used to restore hematopoiesis in transplant patients lacking marrow donors. CB is associated with higher rates of delayed/failed engraftment. Peled et al developed an expansion technology using the copper chelator tetraethylenepentamine (TEPA), which enhanced the expansion of primitive CB populations when combined with early acting cytokines. A phase I clinical trial employing this technology was initiated. 10 patients with high-risk, heavily pre-treated hematologic malignancies (AML-2, ALL-5, HD-2, and NHL-1) have been enrolled with CB units that were cryopreserved in 2 fractions [20:80% (n=2), 40:60% (n=5) or 50:50% (n=3)]. 21days prior to infusion, AC133+ cells were isolated from the smaller (if unequal) or 50% CB fractions using the CliniMACS device and cultured for 21 days in media containing 10% FBS and SCF, FLT-3, IL6, TPO plus the copper chelator TEPA (Gamida). Patients then received myeloablative therapy with ATG and either fludara and busulfan (AML), or fludara, melphalan, thiotepa (ALL, HD, NHL) with infusion of the unmanipulated CB fraction on day 0, and the expanded fraction on day +1. GVHD prophylaxis was methotrex 5 mg/m 2 days 2, 4,7, and tacrolimus for 6 months. The median age was 21 (range 7–51) and weight 69 (range 31–156) kg. The CB units were matched at 4/6 (n=8) or 5/6 (n=2) HLA antigens. The pre-thaw total nucleated cell (TNC) dose of the CB units was a median of 2.5x10 7 /kg with post-thaw TNC of 2.4 x10 7 /kg. Following AC133-selection, the manipulated CB fractions were a median of 73 (range 38–95)% AC133+ with a median of 0.650 (range 0.16–2.7) x10 6 TNCs, which were placed in culture. After 21 days of culture the expanded fraction had 69 (range 2–1638) x10 6 TNCs representing a 207 (range 2–616) fold TNC expansion. Patients received a total (expanded plus unmanipulated) median of 1.8 (range 1.1–6.1) x10 7 TNC/kg and 1.6x10 5 (range 0.4–49.9) CD34+ cells/kg. Two patients have CB cultures in progress. Of the 8 patients transplanted, 1 had autologous recovery with relapse of AML on day 30 and death. Of the remaining patients, 7 were evaluable for neutrophil engraftment and 4 of them for platelet engraftment (2 too early for platelet evaluation and 1 early death). The median time to engraftment was 27 days for neutrophils (range 16–46) and 48 days for platelets (range 27–96). Preliminary analysis suggest a correlation between a shorter time to neutrophil engraftment and total TNC/kg infused (p=0.02), and a trend for CD34+ cells/kg infused (p=0.09). Three patients have developed grade ≤2 acute skin GVHD and one had chronic extensive GVHD of the skin and GI tract; all resolved with steroids. One patient (without GVHD) died of a systemic viral infection on day 56, despite adequate neutrophil recovery (not platelets). All of the remaining patients are all alive and free of malignancy at a median follow-up of 4 (range 1–16) months. Conclusion: There was no toxicity associated with infusion of the TEPA-expanded CB cells. Additional data is necessary to determine the efficacy of this approach. Future directions include the expansion of the entire CB unit and removal of methotrex from the GVHD regimen to improve time to neutrophil engraftment, as well as comprehensive assessment of immune reconstitution.

Procedes de multiplication ex-vivo de cellules progenitrices foetales et / ou adultes et de cellules souches derivees de sang ombilical, de moelle osseuse ou de sang peripherique dans des bioreacteurs pour la greffe de moelle osseuse, la medecine transfusionnelle, la medecine regeneratrice et la therapie genique.

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.

The capacity of the polyamine copper chelator tetraethylenepentamine (TEPA) to induce a marked expansion of human cord blood-derived CD34+ cells was previously demonstrated (Peled T. et al 2002, Br. J. of Haematology 116:655–661). To examine the effect of TEPA also on the ex-vivo expansion of bone marrow (BM)-derived stem cells, we have isolated AC133+ cells from fresh human BM at purities greater than 90% as measured by FACS. Cell cultures (1×104/ml) were expanded in Teflon bags, in the presence of IL-6, TPO, Flt3 ligand and SCF with or without TEPA (5 uM) for 3 weeks and further incubated with cytokines only. Cell types were analyzed by FACS. Total mononuclear cell (TMC) expansion was 1216 ± 176-fold after 5 weeks in TEPA-treated cultures, representing a 2.5- fold higher expansion level as compared to a cytokines-treated control population. 3-weeks old cultures displayed similar amounts of CD34+ and AC133+ cells, irrelevant of treatment. However, following 5 weeks of ex-vivo expansion, TEPA-treated cultures exhibited a significant increase (16.5-fold) in AC133+ cells and in CD34+ cells (105-fold) expansion, as compared to a cytokines-treated control population. In addition, CFU following 5 weeks ex-vivo expansion was 21.4 higher in TEPA-treated cultures, as compared to a cytokines-treated control population. Significantly, the number of AC133+/CD38- cells following 3 weeks ex-vivo expansion was 26-fold, as compared to a cytokines-treated control population. The data presented here strengthen the usefulness of copper chelators to induce proliferation of early progenitors that influence the long term survival and proliferation of early subset populations which are especially reduced in bone marrow as compared to cord blood. The potential of TEPA for increased ex-vivo expansion of long term capacity stem cells derived from sources other than cord blood can provide stem cell-based therapies platform for the autologous treatment of vascular, cardiovascular, diabetes, hepatic, neurodegenerative, skeletal, and other clinical indications.

Calcium and phytate phosphorus were highly soluble below pH 4 at all molar ratios of calcium (Ca) to phytic acid (PA) studied (0.512.67). As pH is increased above 4, there is a drop in solubility, the magnitude of which depends on the Ca:PA molar ratio. Above pH 6, the greatest calcium precipitation occurred at molar ratios between 4 and 6.5; both lower and higher Ca:PA molar ratios showed higher calcium solubility. In contrast, phytate phosphorus solubility decreased in proportion to the Ca:PA molar ratio, showing essentially complete precipitation above the Ca:PA ratio of 5. The pentacalcium phytate salt probably predominates when calcium is not limiting. Under calcium-limiting conditions, the complexes resolubilize as the pH is increased above 7.