Acquisition of CD34 Correlates with Increased Hematopoietic and Self Renewal Activity of CD34−CD133+ Cord Blood Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4143-4143
Author(s):  
Katharina S. Götze ◽  
Matthias Schiemann ◽  
Stefanie Marz ◽  
Christian Peschel ◽  
Robert A.J. Oostendorp
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Division ◽  
Cord Blood ◽  
Blood Cells ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Cord Blood Cells ◽  
Cd34 Expression ◽  
Hematopoietic Activity

Abstract CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.

CD133-enriched CD34− (CD33/CD38/CD71)− cord blood cells acquire CD34 prior to cell division and hematopoietic activity is exclusively associated with CD34 expression

2007 ◽  
Vol 35 (9) ◽  
pp. 1408-1414 ◽  
Author(s):  
Katharina S. Götze ◽  
Matthias Schiemann ◽  
Stefanie Marz ◽  
Volker R. Jacobs ◽  
Gerlinde Debus ◽  
...  
Keyword(s):  
Cell Division ◽  
Cord Blood ◽  
Blood Cells ◽  
Cord Blood Cells ◽  
Cd34 Expression ◽  
Hematopoietic Activity

Cord Blood as a Very Valuable Source of Neonatal Cells but Embryonic-Like Nature Reevaluated

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2897-2897 ◽  
Author(s):  
Anja Buchheiser ◽  
Stefanie Liedtke ◽  
Amelie Pia Houben ◽  
Simon Waclawczyk ◽  
Milaid Stephan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Blood Cells ◽  
Stem Cell Markers ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Human umbilical cord blood has become a very valuable source for hematopoietic transplantation. Our group was able to show that CB contains non-hematopoietic stem cells, which were called unrestricted somatic stem cells (USSCs) with a multipotent differentiation potential. These cells have the potential to differentiate into different germ layers (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). Some studies have now reported a presumably embryonic like nature of cord blood cells. However, Nanog and Oct 4 harbours potential pitfalls for data misinterpretation due to pseudogenes and alternative spliced variants (Liedtke et al. 2007, Liedtke et al. 2008). The related data based on the stem cell markers Nanog and Oct4 concerning these results remain questionable. Therefore, we evaluated the embryonic-like nature of MNCs (n=7), USSC (n=7), CD34+ cells (n=7) derived from cord blood, MNCs from peripheral blood (n=3), MNCs (n=7) and MSCs (n=3) from bone marrow. Using RT-PCR, quantitative RT-PCR and immunohistochemistry, we studied the expression of the pluripotency markers Oct4, Nanog, Sox2 as well as the transcription factors Klf4 and cMyc utilized for the induction of pluripotent stem cells from adult human fibroblasts. The expression level of the transcription factors Klf4 and cMyc was nearly equal in all USSC cell lines and BM MSCs. We neither detected expression of Oct4, Nanog and Sox2 in all tested USSC cell lines nor in MNCs and CD34+ cells from cord blood nor in MSCs, MNCs and CD34+ cells from bone marrow. To increase the sensitivity of our method we performed quantitative Oct4 PCRs. This method revealed that USSCs reach the same Oct4 expression level as human dermal fibroblasts. These results are also supported by the inactive status of the telomerase. As a positive control we used the embryonic carcinoma cell line nTERA-2 showing a high expression of Oct4, Nanog and Sox2. In addition we were able to show that the markers SSEA1, SSEA3 and SSEA4 cannot be used as markers of an embryonic-like phenotype. SSEA-1 recognizes the CD15 epitope, SSEA-4 cross-reacts with an adult MSC subpopulation and SSEA3 was always negative applying the correct isotype controls. However, cord blood does not have to contain embryonic like cells, but it contains neonatal cells as USSC expressing Sox17. For hematopoietic stem cells (HSC) it had already been shown that the transcription factor Sox 17 is required to maintain fetal and neonatal HSC and distinguishes their transcriptional regulation from adult HSCs (Kim et al. 2007). Our results indicate that USSCs and cord blood are neonatal cells without expression of typical embryonic stem cell markers. In more than 10.000 unrelated Cord blood transplants performed so far, no tumor formation associated with an Oct4 positive cell/teratoma formation was observed. Therefore the embryonic-like nature of cord blood cells must be reconsidered.

The Acetylation of AML1-ETO Is Required for Leukemogenesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1588-1588
Author(s):  
Lan Wang ◽  
Alexander Gural ◽  
Fabiana Perna ◽  
Xiaojian Sun ◽  
Xinyang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cord Blood ◽  
Blood Cells ◽  
Target Genes ◽  
Biological Effects ◽  
Self Renewal ◽  
Mouse Leukemia ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 1588 Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare.

Prenatal Origin of Monosomy 7 in Very Young Children.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2557-2557
Author(s):  
Parinda A. Mehta ◽  
Mark Wunderlich ◽  
Teresa Smolarek ◽  
Stella M. Davies ◽  
Sarah Zimmerman ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Young Children ◽  
Cord Blood ◽  
Blood Cells ◽  
Total Sample ◽  
Monosomy 7 ◽  
Very Young Children ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 2557 Monosomy 7 is a common chromosomal abnormality in adults with myelodysplasia (MDS) and AML, seen most frequently in older persons. It is believed to be one of a series of stochastically acquired genetic anomalies that accumulate over many years to complete the process of leukemogenesis. MDS in general and monosomy 7 in particular are rarely seen in children, although cases in very young children are reported. Monosomy 7 arising in very young children likely has a different etiology and may contribute differently to leukemogenesis, as the young age of the children does not allow sufficient time for stochastic acquisition of mutation in the same way as in elderly persons. A proportion of young children with monosomy 7 have an associated DNA instability disorder such as Fanconi anemia (FA) or Dyskeratosis Congenita, and this genetic background likely plays a key role in the evolution of malignancy. In a few reported cases, the occurrence of monosomy 7 is familial, and in a smaller number of cases yet, the clone may resolve spontaneously or wax and wane, as the associated pancytopenia waxes and wanes. We report 5 young children with monosomy 7 in whom we have explored the origins of monosomy 7. In case 1, who presented with MDS and monosomy 7 at 2 years of age, a stored cord blood was available for analysis. The sample was flow sorted and FISH for monosomy 7 was performed on different populations. The monosomy 7 clone was present in approximately 10% of the cord blood cells overall, and the same clone was found in the most primitive population, i.e. CD34+CD38- cells, suggesting in utero origin of this abnormal clone in a very early precursor or stem cell population (Table 1). Similar analyses done on BM or PB samples of 4 additional young patients with monosomy 7, with age range of 4 months to 42 months (3 ½ years)(median 22.5 months), also showed the abnormal clone to be present in CD34+ cells, supporting our hypothesis that monosomy 7 arises in early precursors or stem cells (Table 2, below). In fact, we were able to confirm the presence of monosomy 7 in the CD34+CD38- population in one of these additional cases for which sufficient material was available. Interestingly, in one patient (#4) where we had sequential samplings nine months apart, total BM showed a decrease in the percentage of cells with monosomy 7, from 2.6% to 1.1%. However, in sorted CD34+ cells, we detected a nearly 10 fold increase in cells with monosomy 7 (Table 2). Clinical significance of this finding remains to be determined In summary, our preliminary studies provide proof of concept that in children, the abnormal monosomy 7 clone is present in the hematopoietic stem cell compartment, and can arise in utero in the absence of any known genetic susceptibility syndrome. Table 1. Patient 1 (UCB) FACS Analysis: % of Cord Blood Cells % Monosomy 7 by FISH CD3 (T cells) 23.1% 6/66 (9.1%) CD 19 (B cells) 9.1% 1/44 (0.02%) CD 14 (monocytes) 4% 1/8 (12.5%) CD 16 (granulocytes) 21.5% 0/20 (0%) CD34 1% 70% CD34+CD38- (primitive progenitor) NA 124/136 (91.2%) CD34+38+19-7- (myeloid progenitor) NA 8/13 (61.5%) Table 2 Patient # (BM or PB) % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH % of total sample % Monosomy 7 by FISH CD3 (T cells) CD 19 (B cells) CD 14 (monocytes) CD 16 (granulocytes) CD34+38+ or CD34+ ( progenitor) CD34+CD38- (primitive progenitor) 2 BM 27.0 0.4 1.9 10.9 5.9 46.4 11.3 16.9 1.2 77 (34+) NA NA 3 BM 40.3 2.5 6.6 1.7 2.4 94 15.9 92.4 0.24 78.3 (34+) .01 NA 4-1 BM 37.4 0 13.3 0 3.7 5.9 15.0 3.8 3.9 1.3 1.7 2.8 4-2 BM 13.0 2 9.5 4 0.4 0 35.6 0.4 0.2 12.2 (34+) NA NA 5 PB 21.8 1.5 3.4 NA 22.4 100 9.1 100 NA NA NA NA Disclosures: No relevant conflicts of interest to declare.

Stem Cell Transplantation (Cord Blood Transplants)

Hematology ◽  
2004 ◽  
Vol 2004 (1) ◽  
pp. 354-371 ◽  
Author(s):  
Nelson J. Chao ◽  
Stephen G. Emerson ◽  
Kenneth I. Weinberg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Cell Transplantation ◽  
Blood Cells ◽  
Cord Blood Transplantation ◽  
Adult Patients ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant and non-malignant diseases. However, the ability to identify suitably matched related or unrelated donors can be difficult in some patients. Alternative sources of stem cells such as cord blood provide a readily available graft for such patients. Data accumulated over the past several years have demonstrated that the use of cord blood is an accepted source of stem cells for pediatric patients. Since the cell numbers of hematopoietic progenitors in cord blood is limited and the collection can occur only in a single occasion, its use in adult patients can be more problematic. Here, new developments in the use of cord blood for adults and studies aimed at expansion of cord blood cells and immune reconstitution are described. In Section I, Dr. Nelson Chao describes the early data in cord blood transplantation in adult patients. The patient outcomes are reviewed and analyzed for various factors such as cell dose, HLA typing, and patient selection that could have contributed to the final outcome of these adult patients. Myeloablative as well as nonmyeloablative approaches are presented. Discussion of the various benefits and risks are presented. More recent data from multiple single institutions as well as larger registry data comparisons are also provided. Analyses of these studies suggest methods to improve on the outcome. These newer data should lead to a logical progression in the use of cord blood cells in adult patients. In Section II, Dr. Stephen Emerson describes the historical efforts associated with expansion of hematopoietic stem cells, specifically with cord blood cells. These efforts to expand cord blood cells continue with novel methods. Moreover, a better understanding of stem cell biology and signaling is critical if we are to be able to effectively expand these cells for clinical use. An alternative, more direct, approach to expanding stem cells could be achieved by specific genetic pathways known or believed to support primitive HSC proliferation such as Notch-1 receptor activation, Wnt/LEF-1 pathway induction, telomerase or the Homeobox (Hox) gene products. The clinical experience with the use of expanded cord blood cells is also discussed. In Section III, Dr. Kenneth Weinberg describes immune reconstitution or lack thereof following cord blood transplantation. One of the hallmarks of successful hematopoietic stem cell transplantation is the ability to fully reconstitute the immune system of the recipient. Thus, the relationship between stem cell source and the development of T lymphocyte functions required for protection of the recipient from infection will be described, and cord blood recipients will be compared with those receiving other sources of stem cells. T cell development is described in detail, tracking from prethymic to postthymic lymphocytes with specific attention to umbilical cord blood as the source of stem cells. Moreover, a discussion of the placenta as a special microenvironment for umbilical cord blood is presented. Strategies to overcome the immunological defects are presented to improve the outcome of these recipients.

scholarly journals A Novel Hybrid Transplantation with Autologous Hematopoietic Stem Cells and Matched Unrelated Cord Blood Stem Cells Is Effecttive and Safe for Relapsed or Refractory Lymphoma: A Pilot Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5713-5713
Author(s):  
Yongqian Jia ◽  
Jian Li ◽  
Huan Tao ◽  
Pu Kuang ◽  
Jie Ji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Cell Transplantation ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Refractory Lymphoma ◽  
Cord Blood Cells

Backgroud: Autologous hematopoietic stem cell transplantation (ASCT) is widely recommended for relapsed or refractory lymphoma as an important second-line salvage therapy. Post-transplant relapse is a main issue due to its lacking of the graft versus tumor effects with routine ASCT. Hereby we present a novel hybrid transplantation with autologous stem cells and matched unrelated cord blood cells for relapsed or refractory lymphoma. Method A total of 37 patients with relapsed or refractory lymphoma were enrolled from July 2013 to May 30, 2019 in the West China Hospital of Sichuan University. The autologous peripheral blood stem cells were collected and freezed. HLA matched cord blood cells were searched and provided by the Sichuan Cord Blood Bank. Autologous peripheral blood stem cell transplantation (APBSCT) were infused at day 0 and the selected cord blood cells were infused at day+1 with standard BEAM conditioning regimen. Result The gender distribution was 51.4% female and 48.6% male.The Median age was 37 years old (16-65 years old). The disease characteristics: relapsed or refractory HL 14 cases, relapsed or refractory DLBCL 9 cases, relapsed Burkitt lymphoma 1 case, HGBL with DHL 1 case, DEL 4 cases, Nos 2 cases, DLBCL with high IPI 3 cases. Advanced nasal NK/T cell lymphoma 2 cases, relapdsed EBV-LPD 1 case. The median number of CD34*106/kg for ASCT was 2.35 (1.32-4.58). The median number of total nucleated cord blood cells was 10.2*108 (6.13-17.9) and the CD34+ cord blood cells was 2.72*106 (1.08-5.2). HLA-identical related donor (6/6) was 10.81%, one-antigen-mismatched (5/6) was 72.98%, two-antigen-mismatched (4/6) was 16.21%. All patients were transplanted succesfully with neutrophil recovery of 11days (8-29) and platelet recovery of 14 days (10-120). An early transplanted syndrom with rash or fever were observed in 7 pts (18.9%), while a delayed neutropenia were observed in 5 pts (13.5%). All symptoms were relieved with prednisone therapy. 2 out of 10 pts examined showed sign of microchimerism at 1 month post transplant. With a median 28 months of follow-up (2-73 months), our hybrid transplantation for R/R lymphoma showed that the relapse-free surviaval (RFS) is 90.4% ,and the overall survival (OS) is 86.4%, which is improved remarkablly. The overall OS and RFS were significant different between complete remission (CR) and Non-CR before transplantation (p= 0.002 for OS; p= 0.015 for RFS), but there was no significant difference in the subgroups of HL and NHL. Conclusion This preliminary pilot study suggested that the hybrid stem cell transplantation with autologous stem cells and matched cord blood stem cells is effective and safe for the treatment of high risk lymphoma with limited controlable immuno reactions. Disclosures Zhang: the National Natural Science Foundation of China: Research Funding.

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

2005 ◽  
Vol 12 (3) ◽  
pp. 196-202 ◽  
Author(s):  
Kenichi Teramoto ◽  
Kinji Asahina ◽  
Yuji Kumashiro ◽  
Sei Kakinuma ◽  
Ryoko Chinzei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Hepatocyte Differentiation ◽  
Cord Blood Cells ◽  
Umbilical Cord Blood Cells

Myogenic Potential of Mesenchymal Stem Cells - the Case of Adhesive Fraction of Human Umbilical Cord Blood Cells

2013 ◽  
Vol 8 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Iwona Grabowska ◽  
Wladyslawa Streminska ◽  
Katarzyna Janczyk-Ilach ◽  
Eugeniusz K. Machaj ◽  
Zygmunt Pojda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Myogenic Potential ◽  
Cord Blood Cells ◽  
Umbilical Cord Blood Cells

scholarly journals Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells

2017 ◽  
Vol 57 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Sumie Fujii ◽  
Yasuo Miura ◽  
Masaki Iwasa ◽  
Satoshi Yoshioka ◽  
Aya Fujishiro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Cord Blood Cells ◽  
Umbilical Cord Blood Cells ◽  
Stromal Stem Cells

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

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