Peripheral blood mononuclear cell transplantation to treat no-option critical limb ischaemia: effectiveness and safety

2021 ◽  
Vol 30 (7) ◽  
pp. 562-567
Author(s):  
Nuttawut Sermsathanasawadi ◽  
Kanin Pruekprasert ◽  
Nuttapol Chruewkamlow ◽  
Kulvara Kittisares ◽  
Thanatphak Warinpong ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ankle Brachial Index ◽  
Transluminal Angioplasty ◽  
Critical Limb Ischaemia ◽  
Limb Ischaemia ◽  
Peripheral Blood Mononuclear ◽  
Transcutaneous Oxygen ◽  
Cd34 Cells ◽  
Surgical Bypass

Objective: Local intramuscular transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilised peripheral blood mononuclear cells (PB-MNC) has been shown to be effective for treating patients with no-option critical limb ischaemia (CLI) who are not considered suitable to undergo surgical bypass or percutaneous transluminal angioplasty. The aim of this study was to investigate the effectiveness and safety of PB-MNCs as a treatment for no-option CLI patients. Method: This prospective cohort study was conducted between April 2013 and December 2017. Patients with no-option CLI were treated with G-CSF 5–10 µg/kg/day for 3 days. PB-MNCs (7.1±2.2×10 10 ) with CD34+ cells (2.1±1.2×10 8 ) were collected by blood cell separator and then injected into the calf or thigh of ischaemic limbs. Ankle–brachial index, toe–brachial index and transcutaneous oxygen tension were recorded at 1 and 3 months after injection. The amputation rate and the wound healing rate were also recorded. Results: Eight patients took part in the study. Two patients experienced rest pain relief 1 month after PB-MNC therapy. Five patients had healed ulcer at 6 months after PB-MNC therapy. Limb ischaemia did not improve after PB-MNC therapy in one patient. Below-knee amputation was performed in that patient due to extension of gangrene. Two patients required reinjection of PB-MNCs because of recurrence of ischaemic ulcer. The limb salvage rate after 1 year was 87.5%. Conclusion: Local intramuscular transplantation of G-CSF-mobilised PB-MNCs might be a safe and effective treatment for no-option CLI patients.

Identification Of Specific Hematopoietic Populations Present In Peripheral Blood Mononuclear Cells That Increase Erythroid Output Directly Or Through Feeder/Stromal Cell-Like Properties

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3414-3414
Author(s):  
Esther Heideveld ◽  
Valentina Tirelli ◽  
Francesca Masiello ◽  
Fatemehsadat Esteghamat ◽  
Nurcan Yagci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Cell Contact ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Abstract Hematopoietic development occurs in defined niches that ensure specific interactions and cross-talk with the surrounding stromal cells and different hematopoietic cells themselves. For instance, erythropoiesis occurs on the macrophage island within the bone marrow and the central macrophage is believed to regulate pro-erythroblast differentiation, the final stages of enucleation and reticulocyte maturation. We have observed that the expansion of erythroblasts from total peripheral blood mononuclear cells is increased compared to CD34+ Hematopoietic Stem/Progenitor Cells (HS(P)C) isolated from the same amount of blood [van den Akker, Haematologica, 2010]. This suggests i) the presence of CD34-cells that contribute to erythropoiesis and/or ii) that cell-cell contact or specific secreted growth factors by “helper” cells in these cultures can regulate hematopoiesis/erythropoiesis to increase erythroblast yield. Identifying the specific population(s) underlying the increased erythroid yield and understanding their way of action and regulatory mechanism during HSC differentiation and erythropoiesis is not only important to improve erythroblast culture conditions but may also provide clues to the function of hematopoietic effector cells in the various HS(P)C/erythroblast niches. Using specific lineage depletion (among which CD3 and CD14) we have identified and quantified various human erythroid and non-erythroid CD34+ and CD34- populations on the basis of CD36 co-expression in peripheral blood mononuclear cells (PBMC). Erythroid outgrowth from these CD34- populations and CD34+ populations and their contribution to the total erythroid yield from PBMC was assessed. Interestingly, total erythroid yield from the individual sorted populations did not reach the erythroid yield obtained from total PBMC. We hypothesized that support/feeder cells present in total PBMC are positively influencing in vitro erythropoiesis. In agreement with this, PBMC immuno-depletion of specific hematopoietic cell types identified CD14 cells (monocyte/macrophages) and to a lesser extend CD3 cells (lymphocytes) to be also partly responsible for the increased erythroblast yield. Compared to HS(P)C alone, co-culture of CD14 cells and HS(P)C isolated from PBMC resulted in a 5-10 times increase in CD71high/CD235med erythroblasts. Conditioned medium of CD14 cells as well as transwell experiments reconstituted the effect of the HS(P)C-CD14 co-cultures to 70%-80%, indicating that cell-cell contact plays a minor role. CD14 cells could elicit their effect at different stages during HSPC/HSC differentiation to erythroblasts. Co-culture of CD14 cells with pro-erythroblasts did not increase the cellular yield or proliferation rate. In contrast, two days of CD14 co-culture with CD34+ cells results in a 5 fold increase of total colony forming units without altering the colony lineage dynamics. In agreement with this a 5 fold increase in CD34+ cells was observed. These results indicate that CD14 cells elicit their effect on early hematopoietic progenitors but not on the erythroblast population. The results predict that depletion of CD14+ cells from PBMC should result in a decrease in the total number of CD34+cells. Indeed, we observed a 2 fold decrease of specifically HS(P)Cs and MEPs after two days of culture in PBMCs depleted for CD14 cells. Taken together our data i) identify previously unrecognized erythroid and non erythroid CD34- and CD34+ populations in peripheral blood that contribute to erythroid yield from total PBMC and ii) indicate modulation of HS(P)C outgrowth by specific hematopioietic effector cells present in peripheral blood that can also be found near specific hematopoietic niches in the bone marrow. The involvement of CD3 and CD14 immune cells suggests that HS(P)C and erythropoiesis may be modulated by immune-responses. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 958-970 ◽  
Author(s):  
CE Sandstrom ◽  
JG Bender ◽  
ET Papoutsakis ◽  
WM Miller
Keyword(s):  
Cell Cultures ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Similar Distribution ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

Therapeutic neovascularization by transplantation of mobilized peripheral blood mononuclear cells for limb ischemia

2006 ◽  
Vol 95 (02) ◽  
pp. 301-311 ◽  
Author(s):  
Shu Li ◽  
Bin Zhou ◽  
Zhong Han
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Limb Ischemia ◽  
Doppler Imaging ◽  
Peripheral Blood Mononuclear ◽  
Therapeutic Efficiency ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

SummaryAutolougous transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood mononuclear cells (PBMNCs) improves limb ischemia in patients with arteriosclerosis obliterans of lower extremities and with diabetic foot. However, the mechanism of action of PBMNCs remains elusive. Here, we studied comparatively the effects of the G-CSF-mobilized PBMNCs and CD34-depleted G-CSF-mobilized PBMNCs in an ischemia model of athymic nude mice. Fluorescence-labeled human PBMNCs [1×106] were intramuscularly injected into the unilateral ischemic hindlimbs of mice. Laser Doppler imaging analysis revealed a significantly augmented blood perfusion at day 7, 14 and 28 after operation. The capillary density was also markedly increased and the rate of limb loss was significantly reduced in cell-transplanted groups when compared with those in PBS group. In comparison with G-CSF-mobilized PBMNCs, the therapeutic efficiency of G-CSF-mobilized PBMNCs deprived of CD34+ cells was impaired. Transplanted cells were found to accumulate around arterioles and scatter in capillary networks. Incorporation of transplanted cells into new capillaries was observed in the G-CSF-mobilized PBMNCs group, but was not detected in the group deprived of CD34+ cells. There was an elevated expression of VEGF in ischemic tissue. Colocalization of VEGF and transplanted mononuclear cells within adductor tissue was demonstrated. These findings indicate that G-CSF-mobilized PBMNCs promote vascular growth not only by incorporating into vessel walls but also by supplying angiogenic factors. The depletion of CD34+ cells attenuated the therapeutic efficiency of G-CSF-mobilized PBMNCs in response to ischemia-induced neovascularization.

Stem cell and progenitor cell therapy in peripheral artery disease

2010 ◽  
Vol 103 (04) ◽  
pp. 696-709 ◽  
Author(s):  
Peter Bramlage ◽  
Berthold Amann ◽  
Holger Lawall
Keyword(s):  
Stem Cell ◽  
Cell Therapy ◽  
Peripheral Artery Disease ◽  
Mononuclear Cells ◽  
Ankle Brachial Index ◽  
Tissue Loss ◽  
Critical Limb Ischaemia ◽  
Limb Ischaemia ◽  
Peripheral Artery ◽  
Artery Disease

SummaryAtherosclerotic peripheral artery disease (PAD) is a common manifestation of atherosclerosis. The occlusion of large limb arteries leads to ischaemia with claudication which can progress to critical limb ischaemia (CLI) with pain at rest, and to tissue loss. At present, common therapy for CLI is either surgical or endovascular revascularisation aimed at improving blood flow to the affected extremity. However, major amputation and death are still frequent complications. Exploring new strategies for revascularisation of ischaemic limbs is thus of major importance. Bone marrow (BM)-derived stem and progenitor cells have been identified as a potential new therapeutic option to induce therapeutic angiogenesis. Encouraging results of preclinical studies have rapidly led to several small clinical trials, in which BM-derived mononuclear cells were administered to patients with limb ischaemia. Clinical benefits were reported from these trials including improvement of ankle-brachial index (ABI), transcutaneous partial pressure of oxygen (TcPO2), re-duction of pain, and decreased need for amputation. Nonetheless, large randomised, placebo-controlled, double-blind studies are necessary and currently ongoing (BONMOT-CLI, JUVENTUS and NCT00498069). Further research relates to the optimal cell type and dosage, the isolation method, the role of colony-stimulating factors, administration route, and the supportive stimulation of cells with reduced functioning due to advanced PAD. Autologous stem cell therapy for ischaemic peripheral disease seems to be a promising new tool for the treatment of severe limb ischaemia. Preliminary evidence has established its safety, feasibility and effectiveness on several important endpoints. Several large endpoints studies are underway to further consolidate this evidence.

scholarly journals Thawed and Washed Apheresis Products Keep an Excellent CD34+ Cells Viability for 24 Hours Using Either Voluven or Normal Saline Plus Albumin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5738-5738
Author(s):  
Miguel Blanquer Blanquer ◽  
Carmen Alguero ◽  
Pilar Menchon ◽  
Assumpta Ferrer ◽  
Pilar Martínez Avilés ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Normal Saline ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Time Points ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
The Mean

Abstract Cryopreservation of products rich in progenitor cells is mandatory for the feasibility of autologous hematopietic progenitor cells transplants. The most used product nowadays is the apheresis of mobilized peripheral blood mononuclear cells. However, cryopreservation implies the use of cryoprotectant molecules, such as DMSO, that can be toxic for the patients. Moreover, the thawing of the product goes with some unavoidable cell death and liberation of cytoplasmic content such as cytokines to the medium. And so, adverse reactions during the product infusion are not infrequent. Our group has demonstrated that washing the thawed apheresis products both with Voluven or with normal saline plus 5% albumin (NSA) is able to almost completely avoid infusion reactions without losing CD34+ cells. Here we wanted to know whether it also had the benefit of extending CD34+ viability for 24 hours after washing. METHODS: We thawed 3 spare peripheral blood mononuclear cells apheresis products that had been cryopreserved with 9%DMSO. Ten mL of each product were separated and the remaining volume was splitted in 2 bags to be washed either with Voluven or NSA. Sepax 2 smartwash automatic program was used to wash the cells. The washed cells were stored at 4ºC and a sample was taken and immediately analyzed at 0h, 1h, 2h, 4h and 24h. At the same time points 10 mL of each bag were separated and kept 30 min. at room temperature (RT) before being analyzed. A blood count was performed on all the samples. Flow cytometry was used to measure CD45+ and CD34+ cells viability by 7AAD staining. RESULTS: The mean CD45+ cells viability was 69% after thawing the cells ,81% immediately after washing them with Voluven, and 81%, 80%, 79% and 73% 1h, 2h, 4h and 24h after the wash. When using NSA the mean CD45+ viability was 79%, 92%, 93%, 92%, 93% and 87% at the same time points. When the samples were kept for 30 additional minutes at RT, the mean CD45+ viability was 78%, 81%, 84%, 82%, 84% and 81% with Voluven, and 80%, 92%, 94%, 91%, 95% and 86% with NSA. Regarding CD34+ cells, when washed with Voluven the mean viability was 46%, 87%, 87%, 92%, 83% and 69%, while it was 61%, 91%, 91%, 92%, 89% and 84% when NSA was used. The mean CD34+ cells viability results after 30' at RT for each time point were 79%, 90%, 82%, 91%, 82% and 81% with Voluven and 85%, 90%, 90%, 94%, 93% and 86% with NSA. Moreover the mean viable CD34+ cells recovery at 24h was 90.94% for Voluven and 87,88% for NSA. CONCLUSIONS: We have obtained an excellent stability of the CD34+ cells viability for 24h after washing the mobilized mononuclear cells apheresis products both with Voluven and with NSA when the products are stored at 4ºC. Moreover, the viability is not affected when the cells are kept for an additional 30' at RT. The viable CD34+ cells loss at 24h was scarce. Disclosures Blanquer Blanquer: Pfizer: Research Funding.

scholarly journals Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 958-970 ◽  
Author(s):  
CE Sandstrom ◽  
JG Bender ◽  
ET Papoutsakis ◽  
WM Miller
Keyword(s):  
Cell Cultures ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Similar Distribution ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Abstract Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

CD34+ progenitor cells mobilized by natalizumab are not a relevant reservoir for JC virus

2010 ◽  
Vol 17 (2) ◽  
pp. 151-156 ◽  
Author(s):  
Clemens Warnke ◽  
Vsevolod Smolianov ◽  
Thomas Dehmel ◽  
Marcel Andrée ◽  
Hartmut Hengel ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Jc Virus ◽  
Viral Dna ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells ◽  
Natalizumab Treatment

Background: Progressive multifocal leukoencephalopathy (PML) is associated with natalizumab treatment in patients with multiple sclerosis (MS). It has been hypothesized that natalizumab mobilizes JC virus (JCV)-infected haematopoietic progenitor cells mediating viraemia and subsequently this disease. Objective: The objective of this study was to investigate peripheral haematopoietic progenitor cells for evidence of JCV DNA in MS patients treated with natalizumab. Methods: We assessed JCV and cytomegalovirus (CMV) DNA in magnetically separated CD34+ haematopoietic progenitor cells, peripheral blood mononuclear cells and plasma of 67 natalizumab-treated patients with MS and six PML patients. Results: Viral DNA was not detectable in CD34+ haematopoietic progenitor or peripheral blood mononuclear cells from any sample. Two plasma samples from patients with MS while undergoing natalizumab treatment were JCV-positive. In one case clinically manifest PML developed 8 months thereafter. Conclusions: Our findings do not support the hypothesis that natalizumab mobilizes JC virus-infected CD34+ cells from the bone marrow mediating JC viraemia. Notably, JC viraemia was detected in one patient with MS prior to developing clinical PML. This warrants further study.

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