脐血造血细胞中AC133抗原表达及其功能特征研究

目的 探讨AC133抗原在正常人造血细胞中表达的意义。方法 采用双色直接免疫荧光标记法,使用流式细胞仪测定脐血中造血细胞的免疫表型。通过祖细胞集落形成单位（CFC）和长期培养启动细胞（LTC-IC）的测定对脐血中AC133^ ,CD34^ 和AC133^-CD34^ 细胞的造血潜能进行了研究。结果 脐血单个核细胞（MNCs)中AC133^ 细胞比例为0.67%,AC133^ 细胞中93.4%表达CD34抗原,94.2%为PKH26阳性,虽然三群细胞产生的CFC总数差异无显著性。但AC133^ 细胞中50%以上为粒-单集落形成单位,CD34^ ,AC133^-CD34^ 细胞中60%和80%以上为红系集落形成单位,AC133^ 细胞产生的LTC-IC显著高于CD34^ 和AC133^-CD34^ 细胞。体外培养7d,AC133^ 细胞组细胞总数和CFC分别扩增28.2和7.1倍,6.8%细胞仍保持PKH26阳性。结论 脐血中早期造血干/祖细胞富集在AC133^ 细胞群中,AC133^ 细胞在体外具有较强的增殖能力,既能产生大量的早期定向祖细胞,又能保持一定数量的早期干/祖细胞,AC133抗原可作为临床分选造血干/祖细胞的有效标志。     

AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis

Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2-4 x 10(4)/ml), mononuclear cells (MNC, 1-2 x 10(6)/ml) and AC133negative cells (AC133(neg), 2-4 x 10(4)/ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either 'K36EG'[c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or 'TPOFL' (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133(neg) populations (K36EG FI: 15.04 +/- 5.46; TPOFL FI: 8.59 +/- 2.92, P < 0.05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0.01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20.50 +/- 2.36; TPOFL: 47.00 +/- 7.69; P < 0.05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.     

Flt3L induces the ex-vivo amplification of umbilical cord blood committed progenitors and early stem cells in short-term cultures

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.     

IL-6 precludes the differentiation induced by IL-3 on expansion of CD34+ cells from cord blood

BACKGROUND AND OBJECTIVES: Ex vivo expansion of hematopoietic progenitor cells (HPC) from umbilical cord blood (UCB) is an interesting strategy to obtain a sufficient number of transplantable cells for adults. To define the optimal culture conditions allowing the generation of HPC that retain their proliferative capacity without loss of long-term culture-initiating cells (LTC-IC), the effect of different cytokine combinations on the expansion of CD34+ cells from UCB was assessed. DESIGN AND METHODS: CD34+ cells were cultured in serum-free culture medium with four cytokine combinations: stem cell factor plus thrombopoietin plus flk2/flt3 ligand (STF), STF plus interleukin-3 (IL-3), STF plus interleukin-6 (IL-6) and STF plus IL-6 plus IL-3. After a 1-week culture, the number of CD34+ and CD133+ cells, colony forming units (CFU), LTC-IC and telomerase activity were determined. RESULTS: The addition of IL-6 or IL-3 to the combination of STF significantly enhanced the expansion of CD34+, CD133+ cells and CFU. All cytokine combinations tested induced a slight increase in LTC-IC number except that composed by STF plus IL-3. The greatest induction of telomerase activity was observed with the combination of STF plus IL-3 or plus IL-3 plus IL-6. Decay of the activity along time was observed when the combination of STF plus IL-3 was used, and this effect was reverted by the addition of IL-6. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that the inclusion of IL-6 in a serum-free short-term culture has a beneficial effect on HPC expansion from UCB, and precludes the negative effects induced by IL-3 on LTC-IC expansion and telomerase activity.     

Circulating haemopoietic and endothelial progenitor cells are decreased in COPD.

Circulating CD34+ cells are haemopoietic progenitors that may play a role in tissue repair. No data are available on circulating progenitors in chronic obstructive pulmonary disease (COPD). Circulating CD34+ cells were studied in 18 patients with moderate-to-severe COPD (age: mean+/-sd 68+/-8 yrs; forced expiratory volume in one second: 48+/-12% predicted) and 12 controls, at rest and after endurance exercise. Plasma concentrations of haematopoietic growth factors (FMS-like tyrosine kinase 3 (Flt3) ligand, kit ligand), markers of hypoxia (vascular endothelial growth factor (VEGF)) and stimulators of angiogenesis (VEGF, hepatocyte growth factor (HGF)) and markers of systemic inflammation (tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8) were measured. Compared with the controls, the COPD patients showed a three-fold reduction in CD34+ cell counts (3.3+/-2.5 versus 10.3+/-4.2 cells.microL-1), and a 50% decrease in AC133+ cells. In the COPD patients, progenitor-derived haemopoietic and endothelial cell colonies were reduced by 30-50%. However, four COPD patients showed progenitor counts in the normal range associated with lower TNF-alpha levels. In the entire sample, CD34+ cell counts correlated with exercise capacity and severity of airflow obstruction. After endurance exercise, progenitor counts were unchanged, while plasma Flt3 ligand and VEGF only increased in the COPD patients. Plasma HGF levels were higher in the COPD patients compared with the controls and correlated inversely with the number of progenitor-derived colonies. In conclusion, circulating CD34+ cells and endothelial progenitors were decreased in chronic obstructive pulmonary disease patients and could be correlated with disease severity.     

Evaluation of ex vivo expansion and engraftment in NOD-SCID mice of umbilical cord blood CD34+ cells using the DIDECO "Pluricell System"

The Dideco "Pluricell System" is a commercially available closed device composed of an expansion chamber and a kit of certified reagents that allow haematopoietic stem cell expansion. We have expanded seven umbilical cord blood (UCB) samples following the manufacturer's instructions; two groups of irradiated NOD-SCID mice have been transplanted with expanded and nonexpanded cells from the same UCB, and bone marrow was analysed for the presence of human cells. Average UCB volume was 61.6+/-8.8 ml; mean nucleated cell content was 1090.5+/-189.9 x 10(6). Percentage and number of CD34+ cells were 0.37+/-0.13% and 3.9+/-1.2 x 10(6). After separation, CD34+ cell purity was 82+/-11%. Mean number of inoculated cells was 760 000; mean NC and CD34+ fold expansion at 12 days was 230.4+/-91.5 and 21.0+/-11.9. Both groups of mice showed successful engraftment: the percentage of human cells was higher in the group receiving expanded cells (3.4+/-2.01%) compared to the group receiving nonexpanded cells (1.5+/-0.66%) (P<0.00018, Mann-Whitney test). The cell population obtained after 12 days expansion consisted mainly of myeloid and megakaryocytic progenitors. The CD34+ antigen reached the maximum expression level at day 12 (7.5+/-2.0%). Analysis of lineage-markers for human myelomonocytic, megakaryocytic, B, T, CD34 and erythroid cells, gave evidence that all the lineages were represented in the marrow of transplanted mice.     

Myeloid differentiation and maturation of SCF+IL-3+IL-11 expanded AC133+/CD34+ cells selected from high-risk breast cancer patients

The AC133 antigen is selectively expressed on subset of CD 34+ cells isolated from leukapheresis products from high risk breast cancer patients receiving chemotherapy plus G-CSF. MiniMACS AC133+ isolated cells contained a mean of 85% (80-90) AC133+ cells. Enriched AC133+ cells coexpressed 80% CD34+, 6.6% CD33+ and 2% CD15+. Separated AC133+ cells contained 600 GFU-GM/10(4) cells and 70 BFU-E/10(4) cells. Flow-cytometric analysis indicated that AC133+ cells were isolated from cells population with low granularity (SS), while CD33+ a CD15+ cells had a high granularity. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL11, the expansion of cells increased 19.4 times. The mean percentage of blasts decreased from 100% at the start of culture to 81% on day 3 and 30% on day 7. Promyelocytes were slow to appear with 10% present on day 3, but thereafter increased to 33% on day 7. The appearance of myelocytes and metamyelocytes lagged 3 days behind promyelocytes and continued to increase during culture to become the predominant (30%) cell type on day 7. Very few neutrophils (2%) were observed in any of the cultures on day 7. Monocytes or macrophages were not detected on day 7. By day 7 megakaryocytes were present at low levels (10%). The mean value of CFU-GM in the culture after day 7 of ex vivo expansion in the presence of SCF+IL-3+IL-11 had increased 45-fold, BFU-E 5-fold. After 7 days of expansion with IL-3+SCF+IL-11 cells expressed a mean of 12% CD34+, 8% AC133+, 59% CD33+ and 30% CD15+. The aim of this experiment was to determine whether ex vivo culture of peripheral blood AC133+ cells could generate sufficient numbers of progenitors to potentially abrogate cytopenia after transplantation and passive purging of tumor cells.     

Ex vivo expansion CD34+/AC133+ - selected autologous peripheral blood progenitor cells (PBPC) in high-risk breast cancer patients receiving intensive chemotherapy

AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for engraftment in transplant situations. We studied the effect of stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 11 (IL-11) on the ex vivo expansion of human CD34⁺/AC133⁺ progenitors isolated from leukapheresis products from chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) -mobilized adult donors. MiniMACS AC133⁺ isolated cells contained a mean of 85% (80-90) AC133⁺ cells. Enriched AC133⁺ cells co-expressed CD34⁺ 80%, CD71^low 36.6 % and CD33⁺ 6.6 %. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11, the number of cells increased 19 times. These cells expressed a mean 12% CD34⁺ and 74% CD71⁺ (23% CD 71^high) and 59% CD33⁺. This means that the absolute number of CD34⁺ cells increased slightly, the number of CD33⁺ increased 100 times and cells with high density CD71^high (23%) appeared. These cells represent the cells committed to erythroid differentiation. The increase in the number of CFU-GM with various combinations of cytokines SCF + Il-3 + IL-11 will be discussed. The number of CFU-GM in culture after a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11 increased 45 times.     

The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation

A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34+ cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks. The number of nucleated cells infused per kilogram body weight of the recipient is also reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. To analyze which parameters could be used to evaluate the hematopoietic potential of a UCB graft, we evaluated almost 300 UCB units that were collected for banking for unrelated transplantation. A strong correlation was found between the frequencies of CD34+ cells and the HPC as measured in semi-solid medium cultures. From the various leukocyte subpopulations, the concentration and total numbers of nucleated cells correlated best with both the HPC content and the number of CD34+ cells. Differentiation of these nucleated cells into subsets of leukocytes offered no advantage for better prediction of HPC or CD34+ cells. These results indicate that the nucleated cell count probably reflects the hematopoietic potential of a UCB graft, and may for that reason correlate with the speed of engraftment after transplantation.     

CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants

The success of haematopoietic stem cell (HSC) transplantation largely depends on numbers of transplanted HSCs, which reside in the CD34⁺ populations of bone marrow (BM), peripheral blood stem cells (PBSC) and umbilical cord blood (UCB). More specifically HSCs reside in the CD38^low/? subpopulation, which cannot be objectively discriminated from mature CD34⁺ CD38⁺ progenitors. Thus, better marker combinations for the quantification of more primitive haematopoietic stem and progenitor cells in transplants are required. Recently, by combining CD34 and CD133 we could clearly distinguish CD133⁺ CD34⁺ multipotent and lympho‐myeloid from CD133^low CD34⁺ erythro‐myeloid progenitors in UCB samples. To qualify the assessment of CD133 for routine quality control of adult HSC sources, we analysed the developmental potentials of CD133⁺ and CD133^low subpopulations in BM and PBSC. Similar to UCB, CD133 expression objectively discriminated functionally distinct subpopulations in adult HSC sources. By implementing anti‐CD45RA staining, which separates multipotent (CD133⁺ CD34⁺ CD45RA^?) from lympho‐myeloid (CD133⁺ CD34⁺ CD45RA⁺) progenitor fractions, UCB was found to contain 2–3 times higher multipotent progenitor frequencies than BM and PBSC. To test for the consistency of CD133 expression, we compared CD133⁺ CD34⁺ contents of 128 UCB samples with maternal and obstetrical factors and obtained similar correlations to related studies focusing on CD34⁺ cell contents. In conclusion, implementation of anti‐CD133 staining into existing routine panels will improve the quality control analyses for HSC transplants.     

Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)- 3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.     

Proliferation and differentiation potential of CD133+ and CD34+ populations from the bone marrow and mobilized peripheral blood

CD34 is the most frequently used marker for the selection of cells for bone marrow (BM) transplantation. The use of CD133 as an alternative marker is an open research topic. The goal of this study was to evaluate the proliferation and differentiation potential for hematopoiesis (short and long term) of CD133+ and CD34+ populations from bone marrow and mobilized peripheral blood. Eight cell populations were compared: CD34+ and CD133+ cells from both the BM (CML Ph−, CML Ph+, and healthy volunteers) and mobilized peripheral blood cells. Multicolor flow cytometry and cultivation experiments were used to measure expression and differentiation of the individual populations. It was observed that the CD133+ BM population showed higher cell expansion. Another finding is that during a 6-day cultivation with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), more cells remained in division D0 (non-dividing cells). There was a higher percentage of CD38− cells observed on the CD133+ BM population. It was also observed that the studied populations contained very similar but not the same pools of progenitors: erythroid, lymphoid, and myeloid. This was confirmed by CFU-GM and CFU-E experiments. The VEGFR antigen was used to monitor subpopulations of endothelial sinusoidal progenitors. The CD133+ BM population contained significantly more VEGFR+ cells. Our findings suggest that the CD133+ population from the BM shows better proliferation activity and a higher distribution of primitive progenitors than any other studied population.     

CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation

This study aimed to identify which graft product subset of cells might be the most predictive of late haematopoietic recovery (three to 12 months) following autologous peripheral blood stem cell transplantation (PBSCT). The relationships between the numbers of reinfused CD34+ cells and their immature subsets such as CD34+/CD90+, CD34+/AC133+, CD34+/CD38- and CD34+/HLA-DR- cells, and haemoglobin, white blood cell (WBC) and platelet counts at 3, 6, 9 and 12 months after PBSCT, were studied in 25 patients with haematological and solid malignancies. The total CD34+ cell number, as well as CD34+/CD90+ and CD34+/AC133+ cell numbers, correlated with platelet counts at 3, 6, 9 and 12 months after PBSCT, but the CD34+/CD90+ cells infused best predicted platelet recovery during the first 12 months after PBSCT (P < 0.0238 at any time-point). The CD34+/AC133+ cell dose also correlated with WBC counts at 3 months post PBSCT. In addition, all patients receiving more than 80 x 10(4) CD34+/CD90+ cells/kg showed platelet counts greater than 100 x 10(9)/l at all points after PBSCT, suggesting that this value of the CD34+/CD90+ cells infused was a threshold dose for durable haematopoietic engraftment after PBSCT.     

Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.     

Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

OBJECTIVE: Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ cells from UCB in vitro. MATERIALS AND METHODS: Human placenta MPC was isolated and characterized by morphologic, immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculture with placental MPC. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay. RESULTS: In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin, and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and alpha-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes. In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of cytokines, was significantly enhanced: CD34+ cells by 14.89 +/- 2.32 fold; colony-forming cell (CFC) by 36.73 +/- 5.79 fold; and LTC-IC by 7.43 +/- 2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli. CONCLUSION: These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.     

Human natural killer cell development in a xenogeneic culture system

In vivo and in vitro xenogeneic models have shown the ability of a non-human environment in supporting human haemopoiesis. In the present study, we evaluated the effect of fetal sheep thymic stroma in the in vitro development of natural killer (NK) cells from human haemopoietic progenitors. CD34+HLA-DR+ (CD34+ DR+)Lin- and CD34+DR-Lin- bone marrow (BM) progenitors were cultured for 3 weeks with or without interleukin 2 (IL-2), in fetal sheep thymic stroma contact and transwell cultures. Both progenitors gave rise to NK cells, defined as CD45+CD56+ cells, in the presence or absence of IL-2; however, the percentage of NK cells originated in cultures with IL-2 was significantly higher. Direct contact with stroma seemed to be required for the most immature progenitors, CD34+DR-Lin-, to differentiate along the NK cell lineage. Functional assays revealed that only cells grown in the presence of IL-2 were cytolytic against K562 targets and, curiously, NK cells derived from CD34+DR-Lin- progenitors were more cytotoxic that NK cells derived from CD34+DR+Lin- progenitors. These studies suggest that the ability of fetal sheep thymic stroma in promoting the generation of human NK cells from haemopoietic progenitors may have relevance in terms of NK cell ontogeny and induction of tolerance in transplantation.     

Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells

OBJECTIVE: The regenerative potential of endothelial and hematopoietic progenitor cells in the heart may vary according to their origin. This study was designed to compare the functional effects of CD133+ cells from human cord blood and bone marrow in a mouse model of myocardial injury. METHODS: 5 x 10(5) CD133+ cells from bone marrow (BM(CD133)) or cord blood (UCB(CD133)) were injected in the necrosis border zone of NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice with left ventricular cryoinjury (CI+). Transplanted cells were tracked by immunostaining for hNuclear antigen and by PCR for hDNA. Echocardiography was used to measure contractility. Scar size, capillary density, and cardiomyocyte apoptosis were evaluated by histology. In addition, the myogenic and endothelial differentiation capacity of BM(CD133) and UCB(CD133) was compared in vitro. RESULTS: DNA was detected 4 weeks after cell injection by PCR, but hNuc+ cells were found by immunostaining only after 48 h. Capillary density in both BM(CD133) and UCB(CD133) cell-treated CI+ mice was higher than in control CI+ mice, but not different between BM(CD133) and UCB(CD133) cell-treated hearts. There were no differences in scar size and myocardial mass among BM(CD133), UCB(CD133) and control CI+ mice, but cardiomyocyte apoptosis was reduced by both BM(CD133) and UCB(CD133) cells. The post-injury deterioration of shortening fraction (46.2+/-1% in sham-operated mice and 41.3+/-0.8% in control CI+ mice) was prevented by BM(CD133) cells (45.4+/-0.9%), but not by UCB(CD133) cells (40.8+/-0.7%). On the other hand, both BM(CD133) and UCB(CD133) cells abolished post-injury mortality. In vitro, neither cultivated BM(CD133) or UCB(CD133) cells developed into myocytes, but both readily differentiated towards an endothelial cell phenotype. CONCLUSIONS: While both cord blood and marrow CD133+ cells have some beneficial effects on post-injury angiogenesis and survival, only marrow cells appear to improve myocardial contractility.     

A closed system for the clinical banking of umbilical cord blood

Umbilical cord blood (UCB) is a source of hematopoietic progenitor cells and is used as an alternative to the bone marrow or peripheral blood for treatment of several onco-hematological diseases. Because of the limited number of CD34+ hematopoietic stem cells present in UCB units and of the elevated costs of cryopreservation, it is of paramount importance to select the UCB units that are clinically useful before storage and optimize banking efficiency by designing reliable procedures to process and freeze the selected units. Among the different parameters characterizing UCB, nucleated cell (NC) and CD34+ cell content provides useful criteria to select UCB units since clinical data documented that the infused cell load (both NC and CD34+ cells) plays an important role in the successful outcome of transplants. By evaluating volume, CD34+ cell content, NC total amount, and NC density of 117 UCB units, we found a significant association between CD34+ cell content and NC density and total amount, indicating these parameters as useful to decide UCB clinical utility. Furthermore, we set up a fast procedure to process UCB units for storage. A system for NC separation and volume reduction of UCB samples in a dedicated, germ-free, closed circuit was developed, where plasma and red blood cells (RBC) depletion was obtained by sedimentation in the presence of a 3.5% Polygeline solution. By this separation system, both RBC depletion and high NC and CD34+ cell recoveries were achieved in 60 min, and the yield was comparable to the one obtained by other separation methods. Since Polygeline has been clinically used as a plasma expander and no toxic effects on patients were reported, the protocol can be applied in the large-scale banking of UCB.     

A multifactorial analysis of umbilical cord blood, adult bone marrow and mobilized peripheral blood progenitors using the improved ML-IC assay

OBJECTIVE: Assays that can evaluate the potential of individual human hematopoietic stem cells (HSC) are still lacking. We previously developed the myeloid-lymphoid initiating cell (ML-IC) assay that enumerates single CD34(+) cells that generate long-term culture-initiating (LTC-IC) and NK-initiating (NK-IC) daughter cells, or single primitive progenitors with multilineage potential. When transplanted in vivo, umbilical cord blood (UCB) has greater repopulating ability than bone marrow (BM) or mobilized peripheral blood (MPB). Whether the greater in vivo repopulating ability is due to an increased frequency of HSC in UCB and generative potential of UCB, BM, and MPB CD34(+) cells is not known. MATERIALS AND METHODS: Single UCB, BM, and MPB CD34(+)CD38(-)Lin(-) or CD34(+)CD38(-)CD33(-) cells were plated in ML-IC assay and after 2 to 4 weeks, progeny was evaluated for frequency and generative potential of ML-IC. We also tested whether the ML-IC assay could be used to define if increased numbers of primitive progenitors generated by different cytokines in expansion cultures are mediated by recruitment of quiescent cells or by increasing their generative potential. RESULTS: The frequency of ML-IC in BM, UCB, and MPB was similar, but the generative potential of UCB ML-IC was significantly higher. Substitution of Flt3-L, SCF, and IL-7 with Flt3-L and thrombopoietin significantly increased the generative potential of ML-IC, whereas Flt3-L, SCF, and hyper-IL-6 increased both ML-IC frequency and generative potential. CONCLUSION: The ML-IC assay demonstrates that the greater repopulating ability of UCB is due to the higher generative ability of HSC in UCB. Furthermore, the ML-IC assay can discriminate between cytokine-mediated expansion of hematopoietic progenitors by enhancing generation of immature daughter cells or by recruiting otherwise quiescent cells.     

Transforming growth factor-beta 1 delays formation of granulocyte-macrophage colony-forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haemopoietic progenitor cells

Ex vivo culture and expansion of autologous haemopoietic transplants has been developed to improve tumour cell purging and accelerate haemopoietic reconstitution by transplantation of increased progenitor cell numbers. We studied the effect of the negative haemopoietic regulator, transforming growth factor beta-1 (TGF-beta 1) on primitive precursors during ex vivo expansion of CD34⁺ cells. When added directly to methylcellulose colony-forming assays, TGF-beta 1 potently suppressed the development of granulocyte-macrophage colonies from CD34⁺ enriched peripheral blood progenitor cells (80–90% inhibition). In contrast, expansion of total nucleated cells and granulocyte-macrophage colony-forming cells (GM-CFC) from CD34⁺ progenitors in liquid culture in the presence of stem cell factor (SCF), interleukin (IL)-1 beta, IL-3, IL-6 and erythropoietin (EPO) was inhibited to 32–65% of control culture levels within 14 d when TGF-beta 1 was added, and still produced an average 3.3-fold absolute amplification of GM-CFC. The inhibitory effect of TGF-beta 1 on GM-CFC generation was reversed when it was washed out on day 6 of ex vivo expansion cultures, and total numbers of GM-CFC generated from expansion cultures then reached levels of untreated controls by day 16. Long-term bone marrow culture-initiating cell (LTCIC) numbers were preserved, at least at input levels, over a culture period of 14 d both in control and TGF-beta-1-treated expansion cultures. These findings suggest that TGF-beta 1, a cytokine which induces apoptosis or terminal differentiation in a number of malignant cell types, may be added to ex vivo expansion cultures without loss of primitive cells from autologous haemopoietic transplants.