scholarly journals Effect of recombinant erythropoietin in interaction with stromal factors on cord blood hematopoiesis

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3212-3217 ◽  
Author(s):  
T Abe ◽  
Y Takaue ◽  
Y Kawano ◽  
Y Kuroda
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Membrane Filter ◽  
Culture Conditions ◽  
The Other ◽  
Recombinant Erythropoietin ◽  
Other Hand ◽  
Cd34 Cells

To investigate the effect of recombinant erythropoietin (Epo) on primitive human hematopoietic progenitor cells, we cultured cord blood mononuclear cells (CBMNC) and CB CD34+ cells in a Dexter-type long-term culture system (LTC), to which various concentrations of Epo were added at day 0 or 7, with or without direct contact with irradiated allogeneic human marrow stromal layers. In regular stroma-contact cultures, when CBMNC were inoculated, the addition of Epo at 1 to 10 U/mL induced a significant increase in LTC-initiating cells (LTC-IC), particularly in the myeloid component, compared with the control without Epo. Significantly more LTC-IC were generated by the delayed addition of Epo on day 7 than on day 0. On the other hand, when CD34+ cells were inoculated, physiologic concentrations of Epo (0.1 U/mL) induced a more than twofold increase in LTC-IC, which was attributed equally to both the myeloid and erythroid lineages, only when added on day 0. In stroma-noncontact cultures, which were created using a Transwell 0.4-micron microporous membrane filter, dose-dependent suppression of the myeloid component of LTC-IC was observed with a higher concentration of Epo (1 to 100 U/mL) when CBMNC was inoculated. On the other hand, without Epo, fourfold more LTC-IC was generated from CD34+ cells in stroma-noncontact than in stroma-contact cultures, which was then significantly augmented by the addition of Epo (0.1 or 10 U/mL) on day 0. This increase was due to both the myeloid and erythroid lineages. A higher concentration of Epo (100 U/mL) resulted in a decrease in LTC-IC, mainly in myeloid progeny, in all of the culture conditions. Hence, Epo may play an important physiologic role in the maintenance and proliferation of immature stem/progenitor cells, in close interaction with factors from marrow stromal cells.

Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4954-4954
Author(s):  
Ronald L. Brown ◽  
J. Zhang ◽  
L. Qiu ◽  
A. Nett ◽  
G. Almeida-Porada ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Cell Populations ◽  
Multilineage Differentiation ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Cytokine Combination ◽  
Cells Cultured

Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.

Constitutive Expression of Pax5 in Cord Blood Progenitor Cells Rather Inhibits B Lymphopoiesis as Well as Myelopoiesis through the Exon 9-Dependent and Independent Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2718-2718
Author(s):  
Rieko Sekine ◽  
Toshio Kitamura ◽  
Takashi Tsuji ◽  
Arinobu Tojo
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Constitutive Expression ◽  
Lymphoid Cells ◽  
The Other ◽  
B Lymphopoiesis ◽  
Cd34 Cells ◽  
Independent Mechanism ◽  
Exon 9

Abstract Pax5 is a key regulator of B cell commitment and is indispensable throughout the early stages of B cell differentiation. Human B lymphopoiesis can be simulated by cultivating cord blood (CB) progenitor cells on the murine bone marrow stromal cell line such as HESS-5. When CB CD34+ cells are seeded onto HESS-5 cells and supplemented with SCF and G-CSF, the number of non-adherent myeloid cells initially increases, reaching a maximum during the 3rd week, and decreases rapidly thereafter. On the other hand, foci of small B-lymphoid cells beneath the stromal layer appear during the 2nd week and increase in size until the 6th week. In this culture system, we have identified two C-terminal splice variants of Pax5; one skips exon 9 without subsequent frameshift (del9), and the other has a frameshift insert between exon 8 and 9, resulting in novel C-terminal sequences (ins8′). By quantitative RT-PCR analysis, del9 mRNA was barely detected in CB CD34+ cells and thereafter upregulated in progenitor B cells during the culture. In contrast, wild-type Pax5 (wt) and ins8′ mRNA could not be detected until the 3rd week. Then, wt mRNA was rapidly upregulated 100-fold or more abundantly than del9 and ins8′ mRNA (Fig. 1). Transactivation potential of Pax5 isoforms was examined with a reporter plasmid containing high-affinity Pax5 binding sites from the CD19 gene linked to the minimal TATA-like promoter. As a result, del9 showed constantly higher (up to 3.5-fold) activity, but ins8′ had slightly lower activity than wt Pax5. Furthermore, TLE4, a Groucho co-repressor family member and a known Pax5 partner, inhibited the transcriptional activity of wt Pax5 but not del9 or ins8′ isoform, suggesting its interaction with the exon 9 sequences of Pax5. We next examined the impact of Pax5 on growth and differentiation of CB CD34+ cells by retroviral transduction of each isoform as well as EGFP. After infection, sorted EGFP + cells were subjected to the colony-forming assay and the co-culture assay on HESS-5 cells. Erythroid or mixed colony formation was almost completely inhibited by either isoform. GM-colony formation was moderately inhibited by del9 and ins8′ isoforms, but profoundly suppressed by wt Pax5. In coculture with HESS-5 cells, the number of non-adherent myeloid cells was reduced by Pax5 isoforms in a similar manner to the results of GM-colony formation. Unexpectedly, after 6 weeks of culture, the number of CD19+ adherent lymphoid cells decreased more profoundly in the culture transduced with wt Pax5 than those with del9 and ins8′ isoforms (Fig 2). These results suggest that constitutive expression of Pax5 in cord blood progenitor cells substantially inhibits not only myelopoiesis but also B lymphopoiesis through the exon 9-dependent and independent mechanism. We also raise the possibility that developmentally regulated concentration of Pax5 isoforms may be crucial for human B lymphopoiesis. Figure Figure

Expression of CD10 and CD7 Defines Distinct In Vivo Lymphoid Reconstituting Capacity within Human Cord Blood CD34+CD38-Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3184-3184
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Leonard D. Shultz ◽  
Noriyuki Saito ◽  
Mitsuhiro Fukata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Human Cord Blood ◽  
Cd34 Cells

Abstract Human cord blood (CB) CD34+ cells are known to contain both long-term hematopoietic stem cells (LT-HSCs) and lineage-restricted progenitor cells. In the past, in vitro studies suggested that CD10, CD7 or CD127 (IL7Ra) could be candidate surface markers that could enrich lymphoid-restricted progenitor cells in human CB CD34+ cells (Galy A, 1995, Immunity; Hao QL, 2001, Blood; Haddad R, 2004, Blood). However, in vivo repopulating capacity of these lymphoid progenitors has not been identified due to the lack of optimal xenogeneic transplantation system supporting development of human T cells in mice. We aim to identify progenitor activity of human CB CD34+ cells expressing CD10/CD7 by using newborn NOD-scid/IL2rgKO transplant assay that can fully support the development of human B, T, and NK cells in vivo (Ishikawa F, 2005, Blood). Although LT-HSCs exist exclusively in Lin-CD34+CD38- cells, not in Lin-CD34+CD38+ cells, CD10 and CD7 expressing cells are present in Lin-CD34+CD38- cells as well as in Lin-CD34+CD38+ cells (CD10+CD7+ cells, CD10+CD7- cells, CD10-CD7+ cells, CD10-CD7- cells accounted for 4.7+/−2.7%, 10.5+/−1.9%, 7.6+/−4.4%, and 77.1+/−5.2% in Lin-CD34+CD38- CB cells, respectively). We transplanted 500–6000 purified cells from each fraction into newborn NOD-scid/IL2rgKO mice, and analyzed the differentiative capacity. CD34+CD38-CD10-CD7- cells engrafted long-term (4–6 months) in recipient mice efficiently (%hCD45+ cells in PB: 30–70%, n=5), and gave rise to all types of human lymphoid and myeloid progeny that included granulocytes, platelets, erythroid cells, B cells, T cells, and NK cells. Successful secondary reconstitution by human CD34+ cells recovered from primary recipient bone marrow suggested that self-renewing HSCs are highly enriched in CD34+CD38–CD10–CD7- cells. CD10–CD7+ cells were present more frequently in CD34+CD38+ cells rather than in CD34+CD38- cells. Transplantation of more than 5000 CD34+CD38+CD10–CD7+ cells, however, resulted in less than 0.5% human cell engraftment in the recipients. Within CD34+CD38–CD10+ cells, the expression of CD7 clearly distinguished the distinct progenitor capacity. At 8 weeks post-transplantation, more than 70% of total human CD45+ cells were T cells in the CD10+CD7+ recipients, whereas less than 30% of engrafted human CD45+ cells were T cells in the CD10+CD7– recipients. In the CD10+CD7- recipients, instead, more CD19+ B cells and HLA–DR+CD33+ cells were present in the peripheral blood, the bone marrow and the spleen. Both CD34+CD38–CD10+CD7+ and CD34+CD38–CD10+CD7- cells highly repopulate recipient thymus, suggesting that these progenitors are possible thymic immigrants. Taken together, human stem and progenitor activity can be distinguished by the expressions of CD7 and CD10 within Lin-CD34+CD38- human CB cells. Xenotransplant model using NOD-scid/IL2rgKO newborns enable us to clarify the heterogeneity of Lin-CD34+CD38- cells in CB by analyzing the in vivo lymphoid reconstitution capacity.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

Nicotinamide Modulates Ex-Vivo Expansion of Cord Blood Derived CD34+ Cells Cultured with Cytokines and Promotes Their Homing and Engraftment in SCID Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 725-725 ◽  
Author(s):  
Tony Peled ◽  
Sophie Adi ◽  
Iddo Peleg ◽  
Noga G. Rosenheimer ◽  
Yaron Daniely ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Scid Mice ◽  
Cell Phenotype ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Nicotinamide (NA) is a non-competitive inhibitor of NAD(+)-dependent ADP-ribosyl transferases, of CD38 NADase (a major regulator of cellular NAD levels) and of Sir2 histone-deacetylase. These enzymes are playing a pivotal role in regulation of signal transduction pathways and gene expression. In the present study, we evaluated the effect of NA on the ex-vivo expansion of cord-blood (CB) derived CD34+ cells and their bone-marrow (BM) homing and engraftment potential. Culturing of CD34+ cells for three weeks in the presence of cytokines (SCF, TPO, IL-6, FLT3-ligand) only or cytokines + NA (5mM) resulted in similar expansion of CD34+ cells (40-fold relative to input). However, a remarkable increase in the fraction of CD34+ cells displaying an early progenitor cell phenotype (CD34+Lin−) was observed in the NA-treated cultures as compared with cytokines-only treated cultures (18.6 ± 3% and 0.7 ± 0.06%, n=6, p<0.05, respectively). Tracking the cell-cycle history by PKH2 staining showed fewer division cycles of CD34+ cells cultured with NA. These results may suggest a direct correlation between the rate of proliferation and expansion of CD34+Lin− cells. NA-treated CD34+ cells express similar levels of CXCR4 but display increased migratory activity in response to CXCL12 over CD34+ cells treated with cytokines only (36 ± 19% and 11 ± 4%, n=4, p<0.05, respectively). In order to test their homing potential, similar number of mononuclear cells (MNC), before or following expansion with or without NA, were labeled with CFSE and transplanted into irradiated NOD/SCID mice. Twenty-four hours later the numbers of human cells (CD45+CFSE+) and human progenitor cells (CD34+CFSE+) in the BM were counted. Homing of CD45+CFSE+ cells was comparable in the three groups tested. However, CD34+CFSE+ cells with BM homing potential were 3-fold more numerous in NA-treated cultures relative to cytokines-treated cultures, and 6-fold more than in non-cultured CB cells (n=14, p<0.05). To evaluate engraftment, SCID mice were transplanted with 3x103, 6x103 and 12x103 non-cultured CD34+ cells or their entire progeny following 3-week expansion with cytokines only or cytokines + NA (n = 63). The frequency of SCID repopulating cells (SRC) was estimated by limiting dilution analysis as 1/ 36,756 (non-cultured), 1/19,982 (cytokines), 1/ 2,620 (NA) (SCID engraftment was considered as ≥0.5% human CD45+ cells). We found that, in correlation with homing, NA-treated cells have a 14- and 7.6-fold more SRC than non-cultured cells or cytokine-treated cells, respectively. The marked increase in SCID engraftment potential following culturing with NA may be attributed to both improved homing of CD34+ cells as well as higher proportion of early progenitor cells within the CD34+ cell compartment. Despite their numerical expansion, progenitor cells generated in cytokine-supplemented cultures have reduced homing and engraftment capacity. Our study demonstrates that NA modulates in-vitro expansion and augments the in-vivo homing and engraftment of CB-derived CD34+ cells cultured with cytokines.

scholarly journals Long-term culture of chronic myelogenous leukemia marrow cells on stem cell factor-deficient stroma favors benign progenitors

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1306-1312 ◽  
Author(s):  
R Agarwal ◽  
S Doren ◽  
B Hicks ◽  
CE Dunbar
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Chronic Myelogenous Leukemia ◽  
Stem Cell Factor ◽  
Culture Conditions ◽  
The Other ◽  
Myelogenous Leukemia ◽  
Lower Percentage ◽  
Long Term Culture

Long-term culture of marrow from patients with chronic myelogenous leukemia (CML) has been reported to favor the outgrowth of bcr/abl- progenitor cells in some patients. We examined the effect of the presence of soluble or transmembrane forms of stem cell factor (SCF) in long-term cultures of CML marrow. CD34-enriched cells from CML patients in advanced chronic phase or accelerated phase were plated on immortalized fetal liver stromal cells from homozygous SCF-deficient SI/SI mice (SI/SI4) with or without the addition of soluble human SCF, SI/SI4 cells expressing high levels of the transmembrane form of human SCF (SI/SIh220), or primary human allogeneic stroma. Cells were removed from cultures and plated weekly in colony assays. The clonagenic cell output from cultures completely lacking SCF was lower over the first 2 to 3 weeks, but by 5 weeks was similar to the clonagenic cell output from the other culture conditions. Analysis of bcr/abl transcripts from individual colonies showed a lower percentage of malignant progenitors present in long-term cultures completely deficient in SCF than under the other culture conditions, particularly compared with primary human stroma-containing long-term cultures. SCF may specifically favor malignant versus benign progenitor cells present in the marrow of CML patients, and an abnormal proliferative response to SCF in very primitive cells may be an underlying defect in the pathophysiology of this disease.

scholarly journals Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

2007 ◽  
Vol 16 (6) ◽  
pp. 579-585 ◽  
Author(s):  
Guo-Ping Huang ◽  
Zhi-Jun Pan ◽  
Bing-Bing Jia ◽  
Qiang Zheng ◽  
Chun-Gang Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

Circulating hematopoietic progenitor cells in first trimester fetal blood

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 1967-1972 ◽  
Author(s):  
Cesare Campagnoli ◽  
Nicholas Fisk ◽  
Timothy Overton ◽  
Phillip Bennett ◽  
Timothy Watts ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
First Trimester ◽  
Fetal Life ◽  
Fetal Blood ◽  
Maternal Circulation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The yolk sac and aorto-gonad-mesonephros region are well recognized as the principal sites of hematopoiesis in the developing embryo, and the liver is the principal site of hematopoiesis in the fetus. However, little is known about circulating hematopoietic stem and progenitor cells in early fetal life. We investigated the number and characteristics of circulating progenitors in first trimester blood of 64 human fetuses (median gestational age, 10+4 weeks; range, 7+6-13+6 weeks). CD34+ cells accounted for 5.1 ± 1.0% of CD45+ cells in first trimester blood, which is significantly more than in term cord blood (0.4 ± 0.03%;P = .0015). However, the concentration of CD34+ cells (6.6 ± 2.4 × 104/mL) was similar to that in term cord blood (5.6 ± 3.9 × 104/mL). The total number of progenitors cultured from unsorted mononuclear cells (MNCs) in first trimester blood was 19.2 ± 2.1 × 103/mL, which is similar to that in term cord blood (26.4 ± 5.6 × 103/mL). All lineages were seen: colony-forming unit–GEMM (CFU-GEMM), CFU-GM, BFU-e, BFU-MK, and CFU-MK. Clonogenic assays of CD34+ cells purified from first trimester samples produced mainly two lineages: BFU-e (39.0 ± 9.6 × 103/mL CD34+ cells) and CFU-GEMM (22.6 ± 4.7 × 103/mL CD34+ cells). Short-term liquid culture of first trimester blood MNCs in SCF + IL-3 + Flt-3 (stem cell factor + interleukin-3 + Flt-3) increased, by 7-fold, the numbers of CFU-GEMM and induced a dramatic increase in BFU-e (65.6 ± 12.1–fold). These data show that significant numbers of committed and multipotent progenitors with capacity for expansion circulate in first trimester fetal blood and can be CD34 selected. These cells should be suitable targets for gene transfer and stem cell transplantation and, because fetal hematopoietic progenitors have been demonstrated in the maternal circulation from early gestation, may also be manipulated for noninvasive prenatal diagnosis of major genetic disorders.

scholarly journals Direct comparison by limiting dilution analysis of long-term culture- initiating cells in human bone marrow, umbilical cord blood, and blood stem cells

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3653-3659 ◽  
Author(s):  
R Pettengell ◽  
T Luft ◽  
R Henschler ◽  
JM Hows ◽  
TM Dexter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Long Term Culture ◽  
Limiting Dilution Analysis ◽  
Cd34 Cells ◽  
Limiting Dilution

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.

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