Haematopoietic stem and progenitor cells in human term and preterm neonatal blood.

BACKGROUND AND OBJECTIVES: Whilst cord blood (CB) contains a significant number of haematopoietic stem and progenitor cells suitable for bone marrow transplantation, levels of these cells are very low in the adult circulation. In previous studies, we demonstrated that stem and progenitor cells are present in neonatal blood (NB) and reported the first sibling transplant using a combination of CB and NB for a patient with beta-thalassaemia major. However, our preliminary data showed that the number of CD34+ cells decreased rapidly in the peripheral blood of neonates soon after birth. To further investigate the mechanism of the change of stem and progenitor cells in NB, we measured the steady-state levels of CD34+ cells, early progenitor subsets and the expression of adhesion molecules, in term and preterm neonates. MATERIALS AND METHODS: NB was collected serially from infants at 2, 4, 6, 8, 24 and 48 h after birth and was analysed by three-colour flow cytometry. RESULTS: Our results demonstrated that the number of CD34+ cells rapidly decreased in term NB, particularly during the first 2-6 h of life, by 29.2 +/- 5.55% (P = 0.0003) in absolute counts/ml. A decrease was observed in all subsets of CD34+ cells studied, including the CD33+, CD71+, CD62L+ and CD49d+ populations. In contrast, the CD34+ cell number increased in preterm infants in the first 8 h of life, before starting to decrease. Significant inverse correlations were observed between gestational age and levels of CD34+ cells (P = 0.0065, 4-h collection time-point). CONCLUSION: Our study suggests that changes in the levels of CD34+ stem and progenitor cells in NB might be triggered by parturition and affected by the gestational age. Our findings confirmed that if targeted as a supplementary source of stem and progenitor cells to CB for transplantation, NB should be collected as soon as possible after birth.

Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors.

As an early acting growth factor, flt-3 ligand (FL) promotes the ex vivo expansion of hematopoietic stem and progenitor cells. The effect and mechanism of FL on the development of the megakaryocytic lineage remain unclear. In this study, we compared the effects of FL and stem cell factor (SCF) in combination with other megakaryocyte-promoting cytokines on the differentiation and proliferation of megakaryocytic progenitors and investigated the expression of flt-3 receptors on megakaryocytic cell lines. In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. Using murine bone marrow (BM) cells, we demonstrated that FL at a range of 0-100 ng/ml had no significant mitogenic effect on CFU-MK formation. TPO increased CFU-MK (p < 0.001) but the effect was not significantly modified by FL. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). FL alone did not stimulate megakaryocytopoiesis, possibly due to the lack of receptor expression on megakaryocytes. The effect of FL in augmenting the expansion of CFU-MK in liquid culture might be due to the early action of FL at the pluripotent stem cell stage.

Ex vivo expansion of enriched CD34+ cells from neonatal blood in the presence of thrombopoietin, a comparison with cord blood and bone marrow.

Neonatal blood (NB) contains substantial numbers of stem and progenitor cells which decline rapidly after birth. Using a combination of cord blood (CB) and NB, we performed a successful, sibling transplant for a thalassaemia patient, leading to the proposal that NB could be used as an adjunct to CB for transplantation. This study was aimed at addressing the feasibility of expanding NB and thus minimizing the volume needed from a NB collection. In the presence of early acting cytokines interleukin-1beta (IL-1beta), IL-3, IL-6, stem cell factor (SCF), flt-3 ligand with and without thrombopoietin (Tpo), we compared the expansion capacity of CD34+ enriched cells from CB, NB and bone marrow (BM). Flow cytometry and colony-forming unit (CFU) analyses show that Tpo significantly increased the expansion of CD34+ cells from CB and NB to early and committed progenitors. No significant difference was observed between the expansion of CB and NB at 7, 14 or 21 days of culture in terms of CFU, CD34+ and CD61+ cell subsets. The expansion capacity of BM was significantly lower than that of NB or CB, possibly related to the low proportion of CD34+ CD38- cells observed at day 0. There was a relatively rapid expansion of NB which was evident at day 7 whilst the expansion of CB and BM remained low, suggesting a speedy maturation process in the postnatal infant. The expanded cells, being heterogeneous in their morphology and cell surface marker expression, were mostly of the myeloid lineage (CD45+, CD33+ and HLA-DR+). Our results showed that the expansion capacity of NB is comparable to that of CB and if transplanted, the expanded products of NB might contribute to the engraftment kinetics of the neutrophil and megakaryocyte lineage.

Human neonatal blood: stem cell content, kinetics of CD34+ cell decline and ex vivo expansion capacity.

Haemopoietic stem cells are present in fetal blood but their levels decline rapidly in the peripheral circulation of the infant after birth. We previously reported a case of stem cell transplant in a beta-thalassaemia boy using a combination of the cord blood (CB) and neonatal blood (NB) of his sister. This transplant resulted in a successful engraftment. To investigate the possibility of using NB to supplement CB for related transplants, we further characterized stem and progenitor cells and lymphocyte subsets in 20 NB samples, comparing the findings with those in 20 CB samples. Our data showed that NB contained substantial levels of CD34+ cells, CD34+CD38- cells, colony-forming units-granulocyte macrophage (CFU-GM), colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E) and long-term culture initiating cells (LTCIC). NB was similar to CB in the levels of T lymphocytes, but the amounts of B lymphocytes and natural killer cells were higher in CB (P = 0.033, P= 0.001, respectively). The kinetics of CD34+ cells in NB was investigated in serial blood samples obtained from 10 full-term infants at 2, 4, 6, 8, 24 and 48h after birth. CD34+ cells decreased rapidly after birth, declining to only 30% of the 2h level at 48h (P<0012). The rate of decline was greatest in the first 4 h of life. NB from four infants was expanded by culturing the blood samples in the presence of thrombopoietin (Tpo), interleukin 1beta (IL1beta), IL-3, IL-6, flt-3 ligand and stem cell factor (SCF) for 7 d. This resulted in the increase of CD34+ cells, CFU-GM and CFU-MK by 271+/-179, 556+/-385 and 113+/-75 fold respectively. Three of the five samples expanded for 7 d contained LTCIC. These findings suggest that NB might be a supplementary or alternative source of stem cells to CB for transplant. The ethics and practicality of this approach deserve further exploration.