Isolation and differentiation potential of human mesenchymal stem cells from adipose tissue harvested by water jet-assisted liposuction.

BACKGROUND: In recent years the therapeutic application of extracted adipose tissue for autologous fat grafting and the application of adipose tissue-derived mesenchymal stem cells (adMSC) isolated thereof has progressed. Water-jet assisted liposuction (WAL) is 1 procedure for harvesting adipose tissue and provides a favorable aesthetic outcome combined with high tissue protection. Tissue aspirated by WAL has been successfully applied in grafting procedures. OBJECTIVES: The aims of this study were to confirm the tissue viability and to understand the abundance and mesenchymal differentiation capacity of stem cells within the tissue. METHODS: We analyzed tissue integrity of WAL tissue particles via fluorescence microscopy. The adMSC content was determined by isolating the cells from the tissue. The mesenchymal differentiation capacity was confirmed with cytochemical staining methods. RESULTS: The stromal vascular fraction of WAL tissue showed high viability and contained an average of 2.6 × 105 CD34-positive cells per milliliter of tissue. Thus WAL tissue contains a high number of stem cells. Furthermore adMSC isolated from WAL tissue showed typical mesenchymal differentiation potential. CONCLUSIONS: WAL of adipose tissue is well suited for autologous fat grafting because it retains tissue viability. Furthermore it is a valid source for the subsequent isolation of adMSC with multipotent differentiation potential. LEVEL OF EVIDENCE: 3 Therapeutic.

Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies.

OBJECTIVES: Cell therapy may offer novel therapeutic options for chronic ischemic heart disease. In a clinical trial, we first assessed the feasibility and safety of intramyocardial CD133+ bone marrow cell injection together with coronary artery bypass grafting (CABG). We then tested the hypothesis that CABG plus CD133+ cell injection would result in better contractile function than CABG alone. METHODS: Fifteen patients took part in the safety study, followed by 40 patients who underwent either CABG with cell therapy or CABG alone. Bone marrow was harvested from the iliac crest one day before surgery, and purified CD133+ progenitor cells were injected in the infarct border zone during the CABG operation. LV function was measured by echocardiography and myocardial perfusion by SPECT. RESULTS: In the safety study, no procedure-related complications were observed for up to 3 years. LV injection fraction (LVEF) increased from 39.0% +/- 8.7% preoperatively to 50.2% +/- 8.5% at 6 months and 47.9% +/- 6.0% at 18 months (F = 6.03, P = .012). In the efficacy study, LCEF rose form 37.4% +/- 8.4% to 47.1% +/- 8.3% at 6 months in the group with CABG and cell therapy (F = 24.16, P < .0001) but only from 37.9% +/- 10.3% to 41.3% +/- 9.1% in the CABG-only group (F = 7.72, P = .012). LVEF was significantly higher at 6 months in the group with CABG and cell therapy than in the CABG-only group (P = .03). Similarly, perfusion of the infarcted myocardium improved more in patients treated with CABG and cell therapy than in those treated with CABG alone. CONCLUSION: Intramyocardial delivery of purified bone marrow stem cells together with CABG surgery is safe and provides beneficial effects, though it remains to be seen whether thewe effects produce a lasting clinical advantage.

Dendritic cell subsets in human bronchoalveolar lavage fluid after segmental allergen challenge.

BACKGROUND: Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in human bronchoalveolar lavage fluid (BALF) after allergen challenge are unknown. METHODS: 7 patients with allergic asthma (median 23 years, range 19-25 years) underwent segmental challenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers in BALF and in peripheral blood were analysed using four-colour flow cytometry. RESULTS: Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01-0.08%) and myeloid dendritic cells (mDCs, median 0.47%, range 0.27-0.87%) were detectable in BALF from control segments. CD1a-positive dendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09-1.83%) and mDCs (median 1.82%, range 0.95-2.29%) increased significantly in BALF 24 h (p = 0.018 compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. The percentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by an enhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease in mDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis of dendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less mature phenotype compared with BALF dendritic cells from control segments. CONCLUSION: Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shown for the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen after allergen challenge in patients with asthma.

Methotrexate-albumin and aminopterin-albumin effectively prevent experimental acute graft-versus-host disease.

BACKGROUND: During the last several years, major progress has been made in developing targeted chemotherapy using cytotoxic drugs covalently bound to human serum albumin (HSA). We explored the activity of two antifolates methotrexate (MTX) and aminopterin (AMPT) bound to HSA in prophylaxis and treatment of experimental acute graft-versus-host disease (aGVHD). METHODS: In all, 113 female F1 hybrid BN/Lew-rats were irradiated with 8.2 Gy (n=103) or 9.9 Gy (n=10). One day after irradiation each rat received 4 x 10 bone marrow cells and 1.5 x 10 spleen T-cells from female Lew-rats. GVHD prophylaxis consisted of MTX-HSA 2 mg/kg (n=25), MTX-HSA 0.5 mg/kg (n=8), AMPT-HSA 0.65 mg/kg (n=8), MTX 0.5 mg/kg (n=17), or native HSA (n=39) given intraperitoneally (IP) on days 0, 4, 8, and 12. Treatment of aGVHD consisted of MTX-HSA 2 mg/kg (n=8) or AMPT-HSA 0.5 mg/kg (n=8) given intraperitoneally at the time of onset of aGVHD and subsequently every fourth day (a total of four doses). RESULTS: All animals receiving native HSA developed lethal aGVHD. Prophylactic treatment with MTX-HSA 2 mg/kg prevented aGVHD in 18 of 25 animals and in 6 of 8 receiving AMPT-HSA. In contrast, five out of nine rats receiving free MTX died due to aGVHD. The survival rates in the prophylactic MTX-HSA 2 mg/kg and AMPT-HSA groups were significantly higher compared to the MTX and control groups (P<0.05), while non hematologic toxicity of MTX-HSA was not detectable. AMPT-HSA at a dose of 0.65 mg/kg as well as MTX at a dose of 0.5 mg/kg were associated with temporary weight loss and lethargy. CONCLUSIONS: The albumin conjugates MTX-HSA and AMPT-HSA effectively prevent experimental aGVHD.

Effects of granulocyte-colony-stimulating factor on mobilization of bone-marrow-derived stem cells after myocardial infarction in humans.

Recent experimental studies have shown that granulocyte-colony-stimulating factor (G-CSF) enhanced cardiac function after infarction. The concept of direct cytokine or cell-mediated effects on postischemic myocardial function was tested in the setting of human myocardial infarction subjected to percutaneous coronary intervention. In the FIRSTLINE-AMI study 50 consecutive patients with first ST-elevation myocardial infarction were randomly assigned to receive either 10 microg/kg G-CSF for 6 days after percutaneous coronary intervention in addition to standard medication, or standard care alone. G-CSF administration led to mobilization of CD34(+) mononuclear stem cells (MNC(CD34+)), with a 20-fold increase to 64 +/- 37 MNC(CD34+)/microl at day 6 without significant associated changes in rheology, blood viscosity or inflammatory reaction, or any major adverse effects. At 4 months the G-CSF group showed improved left ventricular ejection fraction of 54 +/- 8% versus 48 +/- 4% at baseline (P <0.001), and no evidence of left ventricular end-diastolic remodeling, with a diameter of 55 +/- 5 mm and improved segmental wall thickening (P <0.001); conversely, in control patients left ventricular ejection fraction was 43 +/- 5% at 4 months (P <0.001), with increased left ventricular end-diastolic dimension of 58 +/- 4 mm (P <0.001), and no segmental wall thickening. In conclusion, the FIRSTLINE-AMI study showed that G-CSF administration and mobilization of MNC(CD34+) after reperfusion of infarcted myocardium may offer a pragmatic strategy for preservation of human myocardium and prevention of remodeling without evidence of aggravated atherosclerosis.

Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI).

BACKGROUND: Considering experimental evidence that stem cells enhance myocardial regeneration and granulocyte colony-stimulating factor (G-CSF) mediates mobilization of CD34+ mononuclear blood stem cells (MNCCD34+), we tested the impact of G-CSF integrated into primary percutaneous coronary intervention (PCI) management of acute myocardial infarction in man. METHODS AND RESULTS: Fifty consecutive patients with ST-segment elevation myocardial infarction were subjected to primary PCI stenting with abciximab and followed up for 6 months; 89+/-35 minutes after successful PCI, 25 patients were randomly assigned in this pilot study (PROBE design) to receive subcutaneous G-CSF at 10 microg/kg body weight for 6 days in addition to standard care, including aspirin, clopidogrel, an ACE inhibitor, beta-blocking agents, and statins. By use of CellQuest software on peripheral blood samples incubated with CD45 and CD34, mobilized MNCCD34+ were quantified on a daily basis. With homogeneous demographics and clinical and infarct-related characteristics, G-CSF stimulation led to mobilization of MNCCD34+ to between 3.17+/-2.93 MNCCD34+/microL at baseline and 64.55+/-37.11 MNCCD34+/microL on day 6 (P<0.001 versus control); there was no indication of leukocytoclastic effects, significant pain, impaired rheology, inflammatory reactions, or accelerated restenosis at 6 months. Within 35 days, G-CSF and MNCCD34+ liberation led to enhanced resting wall thickening in the infarct zone of between 0.29+/-0.22 and 0.99+/-0.32 mm versus 0.49+/-0.29 mm in control subjects (P<0.001); under inotropic challenge with dobutamine (10 microg.kg(-1).min(-1)), wall motion score index showed improvement from 1.66+/-0.23 to 1.41+/-0.21 (P<0.004 versus control) and to 1.35+/-0.24 after 4 months (P<0.001 versus control), respectively, coupled with sustained recovery of wall thickening to 1.24+/-0.31 mm (P<0.001 versus control) at 4 months. Accordingly, resting wall motion score index improved with G-CSF to 1.41+/-0.25 (P<0.001 versus control), left ventricular end-diastolic diameter to 55+/-5 mm (P<0.002 versus control), and ejection fraction to 54+/-8% (P<0.001 versus control) after 4 months. Morphological and functional improvement with G-CSF was corroborated by enhanced metabolic activity and 18F-deoxyglucose uptake in the infarct zone (P<0.001 versus control). CONCLUSIONS: G-CSF and mobilization of MNC(CD34+) after reperfusion of infarcted myocardium may offer a pragmatic strategy for preservation of myocardium and prevention of remodeling without evidence of aggravated restenosis.

Prevention of left ventricular remodeling with granulocyte colony-stimulating factor after acute myocardial infarction: final 1-year results of the Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony-Stimulating Factor (FIRSTLINE-AMI) Trial.

BACKGROUND: Experimental and clinical evidence has recently shown that pluripotent stem cells can be mobilized by granulocyte colony-stimulating factor (G-CSF) and may enhance myocardial regeneration early after primary percutaneous coronary intervention (PCI) management of acute myocardial infarction. Sustained or long-term effects of mobilized CD34-positive mononuclear stem cells, however, are unknown. METHODS AND RESULTS: Thirty consecutive patients with ST-elevation myocardial infarction undergoing primary PCI with stenting and abciximab were selected for the study 85+/-30 minutes after PCI; 15 patients were randomly assigned to receive subcutaneous G-CSF at 10 microg/kg body weight for 6 days in addition to standard care including aspirin, clopidogrel, an angiotensin-converting enzyme inhibitor, beta-blocking agents, and statins. In patients with comparable demographics and clinical and infarct-related characteristics, G-CSF stimulation led to sustained mobilization of CD34 positive mononuclear cells (MNC(CD34+)), with a 20-fold increase (from 3+/-2 at baseline to 66+/-54 MNC(CD34+)/microL on day 6; P<0.001); there was no evidence of leukocytoclastic effects, accelerated restenosis rate, or any late adverse events. Within 4 months, G-CSF-induced MNC(CD34+) mobilization led to enhanced resting wall thickening in the infarct zone of 1.16+/-0.29 mm (P<0.05 versus control), which was sustained at 1.20+/-0.28 after 12 months (P<0.001 versus control). Similarly, left ventricular ejection fraction improved from 48+/-4% at baseline to 54+/-8% at 4 months (P<0.005 versus control) and 56+/-9% at 12 months (P<0.003 versus control and paralleled by sustained improvement of wall-motion score index from 1.70+/-0.22 to 1.42+/-0.26 and 1.33+/-0.21 at 4 and 12 months, respectively), after G-CSF (P<0.05 versus baseline and P<0.03 versus controls). Accordingly, left ventricular end-diastolic diameter showed no remodeling and stable left ventricular dimensions after G-CSF stimulation, whereas left ventricular end-diastolic diameter in controls revealed enlargement from 55+/-4 mm at baseline to 58+/-4 mm (P<0.05 versus baseline) at 12 months after infarction and no improvement in diastolic function. CONCLUSIONS: Mobilization of MNC(CD34+) by G-CSF after primary PCI may offer a pragmatic strategy for improvement in ventricular function and prevention of left ventricular remodeling 1 year after acute myocardial infarction.

Human cord blood cells induce angiogenesis following myocardial infarction in NOD/scid-mice.

OBJECTIVE: We tested the hypothesis that intravenously administered human umbilical cord blood (hUCB) cells contribute to repair processes following myocardial infarction. METHODS: hUCB mononuclear cells containing 0.11% to 1.1% CD34(+) cells were injected in the tail vein of NOD/scid mice that had (MI+) or had not (MI-) previously undergone ligation of the left anterior coronary artery (LAD). Homing to bone marrow and solid organs was determined by polymerase chain reaction (PCR) for human DNA (hDNA) using human-specific primers of Locus D7Z1. Immunostaining was used for phenotypic analysis, and capillary density as well as myocardial scar formation was assessed. Moreover, expression of stromal cell-derived factor-1 (SDF-1) was studied in infarcted and in normal hearts. RESULTS: hDNA was detected in marrow, spleen, and liver of both MI+ and MI- mice 24 h, 1 week, and 3 weeks after cell injection. In the heart, however, hDNA was detected in 10 of 19 MI+ mice but in none of the MI- mice (p=0.002). Infarct size was smaller in cell-treated MI+ mice than in untreated MI+ hearts (38.7 versus 47.8%, P<0.05), and there was also less collagen deposition. In cell-treated MI+ mice, capillary density in the infarct border zone was approximately 20% higher (p=0.03), and clusters of hUCB-derived cells were detected in the perivascular interstitium. Occasionally, chimeric capillaries composed of human and mouse endothelial cells were found, but the vast majority of neo-vessels appeared to consist of mouse cells only. Up to 70% of the cord blood-derived cells in the heart were CD45(+). There was no evidence of cardiomyocyte differentiation as determined by co-localization of HNA or HLA-I with GATA-4 or Connexin 43. In infarcted myocardium, expression of SDF-1 mRNA was approximately 7-fold higher than in normal hearts. CONCLUSIONS: hUCB cells migrate to infarcted, not to normal myocardium, where they engraft, participate in neoangiogenesis, and beneficially influence remodelling processes. Cord blood cells may hence be useful for cell therapy of ischemic heart disease.

Treosulfan and fludarabine: a new toxicity-reduced conditioning regimen for allogeneic hematopoietic stem cell transplantation.

New conditioning regimens are being explored to reduce toxicity and enable allogeneic bone marrow transplantation in patients not eligible for conventional transplantation. We have investigated treosulfan, an alkylating agent, with the aim of developing an efficient and reliable but less-toxic conditioning regimen. A series of 30 patients who were not eligible for standard conditioning therapy received transplants from HLA-matched related (n = 14) or unrelated (n = 16) donors after administration of treosulfan 10 g/m2 intravenously daily for 3 days and fludarabine 30 mg/m2 intravenously daily for 5 days. Patients receiving grafts from unrelated donors also were given rabbit antithymocyte globulin 10 mg/kg intravenously daily for 3 days. All patients achieved prompt neutrophil and platelet recovery. Extramedullary toxicity was generally mild with Common Toxicity Criteria (CTC) grade 3 or 4 attributable to the conditioning seen only with transaminases. Complete donor chimerism was achieved by 90% of the patients. Acute graft-versus-host disease (GVHD) grade III or IV developed in 14% of the patients and chronic GVHD in 39%. An estimated overall survival rate of 73% and an event-free survival rate of 49% have been reached after a median of 22 months (range, 7.4-33.4 months). In summary, the combination of treosulfan and fludarabine is a safe and efficient conditioning regimen.

Lack of influence of omega-3 fatty acid-enriched lipids on apoptosis and secondary necrosis of cultured human lymphocytes.

OBJECTIVE: The anti-inflammatory properties of parenteral nutrition might be improved by enrichment with omega-3 polyunsaturated fatty acids (PUFAs), which are responsible for the enhanced release of metabolites derived from eicosapentaenoic acid. Under physiologic conditions, lymphocyte populations are regulated by cellular mechanisms such as apoptosis. In contrast to cell death by necrosis, apoptosis does not induce an inflammatory response that might injure the host. METHODS: Apoptosis and necrosis of cultured human blood lymphocytes were investigated in vitro after incubation for 48 and 72 h with three lipid emulsions containing 50% medium-chain triacylglycerols. The lipid emulsions differed in the percentage of long-chain triacylglycerols, which were replaced in part by different amounts of omega-3 PUFA (8%, 20%, or 40%). Rates of apoptosis and necrosis of lymphocyte subpopulations were analyzed with a sensitive annexin V flow cytometric assay. RESULTS: After 48 and 72 h of incubation, time- and dose-dependent increases of apoptosis and necrosis, respectively, were found in all lymphocyte subsets regardless of the percentage of omega-3 PUFAs. CONCLUSIONS: Our results suggested that enrichment with omega-3 PUFAs in the tested lipid emulsions does not alter apoptosis and secondary necrosis of lymphocyte populations. Thus PUFAs may exert their functional effects through other mechanisms.

Autologous bone-marrow stem-cell transplantation for myocardial regeneration.

Implantation of bone-marrow stem cells in the heart might be a new method to restore tissue viability after myocardial infarction. We injected up to 1.5x10(6) autologous AC133+ bone-marrow cells into the infarct border zone in six patients who had had a myocardial infarction and undergone coronary artery bypass grafting. 3-9 months after surgery, all patients were alive and well, global left-ventricular function was enhanced in four patients, and infarct tissue perfusion had improved strikingly in five patients. We believe that implantation of AC133+ stem cells to the heart is safe and might induce angiogenesis, thus improving perfusion of the infarcted myocardium. See Commentary page 11

Interferon-alpha inhibits cell cycle progression by Ba/F3 cells through the antagonisation of interleukin-3 effects on key regulators of G(1)/S transition.

The molecular mechanisms of interferon-alpha (IFN-alpha)-mediated cell growth inhibition are incompletely understood. Here, we have analysed how IFN-alpha interferes with the interleukin-3 (IL-3)-stimulated cell cycle progression by Ba/F3 cells. The antiproliferative cytokine caused a delay in cell cycle progression, which correlated with a diminished activation of the cyclin-dependent kinases 2 and 4 in IL-3-stimulated cells. While IFN-alpha did not affect the expression of p27(Kip1) and p21(Waf1), it efficiently inhibited the IL-3-induced expression of D-type cyclin and cyclin E proteins. No IL-3-antagonistic effects of the IFN, however, were observed at the mRNA level of cyclin expression. Furthermore, IFN-alpha suppressed the IL-3-induced release of E2F transcription factors from the retinoblastoma protein (pRb) and enhanced pRb-mediated transcriptional repression. The growth factor-antagonistic action of IFN-alpha correlated with a strong stimulation of protein kinase R expression, suggesting that inhibition of protein synthesis plays a pivotal role in IFN-alpha-mediated inhibition of cell cycle progression.