Development of a Novel NOD/SCID Transplant System That Provides Enhanced Detection of Rapid-SRC and Insight into Their Self-Renewal and Mobilization.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 249-249 ◽  
Author(s):  
Joby L. McKenzie ◽  
Olga I. Gan ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Nk Cells ◽  
Cell Biology ◽  
Scid Mice ◽  
Immune Recognition ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Insight Into

Abstract The conventional NOD/SCID xenotransplant model provides a powerful tool to characterize human hematopoietic stem cells. This system relies on IV injection of transplanted cells, with subsequent circulation through the blood prior to homing to appropriate niches. Two major limitations of this model are the presence of residual host factors that resist engraftment (i.e. NK cells and macrophages) and inability to detect stem cells that are incapable of homing or surviving in the circulation. We previously showed that rapid-SRC (R-SRC) were more efficiently detected by direct intrafemoral (IF) injection compared to IV transplantation (Nat Med 2003). Additionally, others showed that depletion of NK cell activity detects a short-term repopulating cell indicating that immune recognition is also important. R-SRC are found in the Lin-CD34+CD38+/lo population and produce a robust human erythromyeloid graft 2 weeks post-transplant. R-SRC are critical for stem cell therapies that require rapid engraftment and their characterization requires an efficient assay. To determine the role of cellular resistance factors we compared human engraftment in NOD/SCID mice, NOD/SCID-B2 microglobulin-null (NOD/SCID-B2m−/−) mice that are depleted of NK cells, or we administered a neutralizing antibody against the IL-2R B-chain (CD122) to NOD/SCID mice. CD122 depletes several populations including NK cells and macrophages. 4–5 x 104 Lin-CD34+CD38lo cells purified from CB were injected IF or IV into these recipients and human engraftment was determined at 2 weeks post-transplant to assay for R-SRC. In addition to determining engraftment levels, we also used the IF assay to gain insight into migration/mobilization function of R-SRC by examining human engraftment in other bones. Human myelolymphoid (CD45+) engraftment in the injected femur (RF) was significantly higher (p<0.05) in IF injected anti-CD122 treated NOD/SCID mice compared to all other groups. Since IF NOD/SCID-B2M−/ − mice had the next highest engraftment levels, these data indicate that R-SRC are very sensitive to NK activity. However the data clearly show that CD122+ cells also play a significant role in resisting stem cell engraftment. Importantly, CD122+ cells markedly affected R-SRC migration/mobilization since there was significantly higher engraftment in non-injected bones from anti-CD122 treated mice even when compared to the NOD/SCID-B2M−/ − mice. Our previous clonal analysis showed that R-SRC that are found in non-injected bones also self-renew in the injected bone before migration. We conclude that in addition to NK cells, CD122+ cells (likely macrophages) prevent the direct engraftment of R-SRC when delivered by IF or IV injection as well as their subsequent in vivo self-renewal and/or migration. Modification to the standard NOD/SCID assay by IF injection in combination with anti-CD122 provides a powerful tool to identify novel populations of stem cells as well as insight into fundamentally important properties of stem cell biology and transplantation. Mouse (n) Injection Tissue CD45+ (%) Erythroid (CD45-CD36+glyA+ (%) Total * RF-injected rt femur;BM-noninjected lt femur, pelvis, two tibiae;glyA-glycophorinA;*-total CD45+plus CD45-erythroid engraftment NOD/SCID anti-CD122 (15) IF RF 13.2 41.4 54.6 BM 4.9 23.3 28.1 NOD/SCID anti-CD122 (18) IV RF 5.3 26.5 31.8 BM 6.6 33.5 40.1 NOD/SCID (13) IF RF 3.6 11.9 15.5 BM 0.9 2.6 3.5 NOD/SCID (15) IV RF 1.4 3.9 5.3 BM 1.3 4.2 5.5 NOD/SCID/B2M−/ − (6) IF RF 7.2 31.9 39.1 BM 1.8 7.0 8.8 NOD/SCID/B2M−/ − (9) IV RF 3.5 4.9 8.3 BM 3.4 9.4 12.8

SRC within the Lin-CD34+CD38+/Lo Population Possess Heterogeneous Migration, Repopulation, and Self-Renewal Potential.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2670-2670
Author(s):  
Joby L. McKenzie ◽  
Olga I. Gan ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Neutralizing Antibody ◽  
Clonal Analysis ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Primary Mouse ◽  
The Individual ◽  
Insight Into

Abstract The SCID repopulating cell (SRC) xenotransplant assay is a powerful tool for characterizing human hematopoietic stem cells. Injection of hematopoietic cells directly into the intrafemoral (IF) cavity along with injection of a neutralizing antibody against residual murine NK cells and macrophages provides additonal improvements to the method (ASH 2004). IF injection permitted identification of a novel rapid-SRC (R-SRC) within the Lin-CD34+CD38+/Lo population that generated an erythromyeloid graft within 2 weeks post-transplant (Nat Med 2003). We found that this population also provides multi-lineage engraftment at 6 weeks post-transplant raising the question of whether the R-SRC had self-renewal potential. Lentivector-mediated clonal tracking was used to determine the self-renewal capacity of the individual cells within the Lin-CD34+CD38+/Lo population. Clonal analysis in primary recipients injected by IF with 5 x 10e4 Lin-CD34+CD38+/Lo and analyzed at 6 weeks showed that a subset of clones present in the injected femur were found in other bones, indicating that some individual SRC had self-renewed in the injected femur and migrated to other hematopoietic tissues. To directly test for self-renewal of migrating and non-migrating SRC, the original injected femur and the other bones (non-injected femur, two tibias and the pelvis) from each primary mouse was injected by IF into two individual secondary mice, respectively. 1) At 6 weeks post-transplant, each cell source produced substantial secondary grafts establishing that the Lin-CD34+CD38+/Lo population contains SRC with self-renewal potential. 2) Clonal analysis revealed heterogeneous self-renewal properties of individual SRC found in the primary mice; some made major contributions to all hematopoietic territories of secondary mice while others did not engraft secondary mice. 3) Interestingly, in some cases clones were detected in secondary mice that had been below detection in the primary mouse, suggesting that upon transplant into primary mice the SRC either did not divide or if they divided they returned to quiescence. Secondary transplantation was a stimulus for their activation to produce a graft of differentiated progeny. 4) Cases were observed where an active clone was found in a secondary mouse (transplanted from the primary injected femur) that had been below detection within the primary injected femur. However, the non-injected bones from this primary mouse as well as secondary mice derived from these bones all contained that same clone. We conclude that upon IF injection this SRC underwent self-renewal divisions and some of these progeny migrated to other bones and established a graft and also self-renewed, while in the injected bone the SRC likely returned to quiescence, only to be reactivated by secondary transplant. 5) Evaluation of two secondary recipients derived from one primary injected femur at 3 weeks when the graft is mainly myeloerythroid and at 6 weeks when it is mainly B cell and myeloid demonstrated that different lineage compositions were initiated by the same stem cell. The combination of clonal marking and IF injection provides an unprecedented insight into the earliest steps of stem cell function following transplantation. Although our clonal analysis is ongoing, it appears that rapid self-renewal and migration following IF injection represents a hallmark of a primitive subclass of SRC. It is essential to gain insight into the complex composition of the human stem cell compartment to develop effective stem cell-based therapies.

Superior Impact of p18INK4c over p27kip1 on Long-Term Repopulating Ability of Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 796-796
Author(s):  
Hui Yu ◽  
Hongmei Shen ◽  
Xianmin Song ◽  
Paulina Huang ◽  
Tao Cheng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract The G1-phase is a critical window during the cell cycle in which stem cell self-renewal may be balanced with differentiation and apoptosis. Increasing evidence suggests that the cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1/Waf1, p27kip1, p16INK4A, and p18INK4C (p21, p27, p16 and p18 hereafter) are involved in stem cell self-renewal, as largely demonstrated in murine hematopoietic stem cells (HSCs). For example, we have recently demonstrated a significant increase of HSC self-renewal in the absence of p18 (Yuan et al, Nature Cell Biology 2004). But the actual roles of these CKIs in HSCs appear to be distinct as p21 and p18 have opposite effects (Yu H et al, ASH 2004) whereas p16 has a limited effect (Stepanova et al, Blood 2005) on HSC exhaustion after serial bone marrow transfer. Like p18, however, p27 was recently reported to also inhibit HSC self-renewal due to the fact that the competitive repopulating units (CRUs) were increased in p27−/− mouse bone marrow (Walkley et al, Nature Cell Biology 2005) in contrast to the results in a previous report (Cheng T et al, Nature Medicine 2000). To further gauge the impact of p18 versus p27 on the long-term repopulating ability (LTRA) of HSCs, we have generated different congenic strains (CD45.1 and CD45.2) of p18−/− or p27−/− mice in the C57BL/6 background, allowing us to compare them with the competitive repopulation model in the same genetic background. The direct comparison of LTRA between p18−/− and p27−/− HSCs was assessed with the competitive bone marrow transplantation assay in which equal numbers of p18−/− (CD45.2) and p27−/− cells (CD45.1) were co-transplanted. Interestingly, the p18−/− genotype gradually dominated the p27−/− genotype in multiple hematopoietic lineages and p18−/− HSCs showed 4-5 times more LTRA than p27−/− HSCs 12 months after cBMT. Further self-renewal potential of HSCs was examined with secondary transplantation in which primarily transplanted p18−/− or p27−/− cells were equally mixed with wild-type unmanipulated cells. Notably, while the p18−/− cells continued to outcompete the wild-type cells as we previously observed, the p27−/− cells did not behave so in the secondary recipients. Based on the flow cytometric measurement and bone marrow cellularity, we estimated that transplanted p18−/− HSCs (defined with the CD34−LKS immunophenotype) had undergone a 230-fold expansion, while transplanted p27−/− and wild-type HSCs had only achieved a 6.6- and 2.4-fold expansion in the secondary recipients respectively. We further calculated the yield of bone marrow nucleated cells (BMNCs) per HSC. There were approximately 44 x 103, 20.6 x 103, and 15 x 103 BMNCs generated per CD34−LKS cell in p18−/−, p27−/− and wild-type transplanted recipients respectively. Therefore, the dramatic expansion of p18−/− HSCs in the hosts was not accompanied by decreased function per stem cell. Our current study demonstrates that hematopoietic engraftment in the absence of p18 is more advantageous than that in the absence of p27, perhaps due to a more specific role of p18 on self-renewal of the long-term repopulating HSCs.

Point: AML Stem Cells are Immature, Rare, and Targets for Curative Therapy in AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-34-SCI-34
Author(s):  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Research Funding ◽  
Residual Disease ◽  
Human Experimentation ◽  
Term Survival ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cellular Hierarchy

Abstract Abstract SCI-34 The cellular and molecular basis for the heterogeneity that exists within the individual cells that make up a tumour is not well understood. There is clear evidence from the historical literature of human experimentation, the vast majority of leukemia blasts are post-mitotic and generated from a rapidly proliferating fraction of <5% of total leukemic blasts. These same studies also established that a rare subfraction of blasts were quiescent and only entered cell cycle in the order of weeks or months. The cancer stem cell (CSC) model postulates that such heterogeneity arises because the tumour is organized as a cellular hierarchy sustained by a CSC at the apex. If a tumour is homogeneous the CSC model would not apply. By employing the principles of stem cell biology, first worked out in hematopoiesis, of clonal assays and prospective cell purification, there is solid evidence that human AML follows a CSC model. LSCs are the only AML cells capable of self-renewal while still generating rapidly proliferating progenitors and terminal leukemic blasts. Our studies have also demonstrated that LSCs are not functionally homogeneous but, like the normal hematopoietic stem cell (HSC) compartment, comprised of distinct hierarchically arranged LSC classes. Distinct LSC fates derive from heterogeneous self-renewal potential. Finally, many LSC appear to be quiescent. Thus, the AML clone is organized as a hierarchy that originates from LSC, which produce AML-CFU and leukemic blasts. AML is organized as a cellular hierarchy sustained by LSCs at their apex. The rare occurrence, generally dormant nature, and abnormal apoptotic response of LSCs are all properties that may render them resistant to conventional chemotherapeutics that target proliferating cells. In addition, minimal residual disease occurrence and poor survival have been attributed to high LSC frequency at time of diagnosis in AML patients. Although there are a number of well known problems with xenotransplant assays, recent improvements to this model have been employed to assess both the frequency of LSC in a cross section of AML samples as well as the nature of the cell surface phenotype of the LSC. Collectively this research has shown that the LSC frequency can vary over many orders of magnitude from 1% to <1 per 106, with some genetic AML subtypes such as MLL leukemias possessing the highest LSC frequency. Thus even with the most optimized assay system they represent a rare subfraction of AML blasts for most forms of leukemia. The properties of AML-LSC make them especially resistant to standard cytoablative therapeutics, so LSC-targeted therapies are clearly needed to improve long term survival for this dismal disease. A number of new therapeutics are being developed and evaluated in the xenotransplant assays and several are now moving into clinical trial. With translation of lab-based research into the clinic, the next several years will begin to provide definitive answers to the most vexing question the continues to surround the whole field of cancer stem cells: while the importance of CSC are clearly seen in numerous xenotransplant models of leukemic and solid tumours, how relevant are they in the human disease? Dick JE. Stem cell concepts renew cancer research. Blood 2008;112:4793-807. Disclosures Dick: Roche: Research Funding; CSL Ltd: Research Funding.

scholarly journals Human NK Cells in Autologous Hematopoietic Stem Cell Transplantation for Cancer Treatment

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1589
Author(s):  
Ane Orrantia ◽  
Iñigo Terrén ◽  
Gabirel Astarloa-Pando ◽  
Olatz Zenarruzabeitia ◽  
Francisco Borrego
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Cell Biology ◽  
Nk Cell ◽  
Hematopoietic Stem ◽  
Autologous Hsct

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes with the ability to recognize and kill malignant cells without prior sensitization, and therefore, they have a relevant role in tumor immunosurveillance. NK cells constitute the main lymphocyte subset in peripheral blood in the first week after hematopoietic stem cell transplantation (HSCT). Although the role that NK cells play in allogenic HSCT settings has been documented for years, their significance and beneficial effects associated with the outcome after autologous HSCT are less recognized. In this review, we have summarized fundamental aspects of NK cell biology, such as, NK cell subset diversity, their effector functions, and differentiation. Moreover, we have reviewed the factors that affect autologous HSCT outcome, with particular attention to the role played by NK cells and their receptor repertoire in this regard.

scholarly journals Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2460-2460 ◽  
Author(s):  
Hairui Su ◽  
Szu-Mam Liu ◽  
Chiao-Wang Sun ◽  
Mark T. Bedford ◽  
Xinyang Zhao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Arginine Methylation ◽  
Poly I:C ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Arginine Methyltransferase ◽  
Hematopoietic Stem

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Maintenance of Earlier Hematopoietic Stem Cell (HSC) Phenotype and Improved NOD/SCID Engraftment for UCB Expanded Short-Term in Cytokines over a Human Mesenchymal Stem Cell (huMSC) Platform.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2846-2846
Author(s):  
M. Kozik ◽  
J. Banks ◽  
L. Fanning ◽  
M. Finney ◽  
Y. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Flow Analysis ◽  
Scid Mice ◽  
Mouse Bone Marrow ◽  
Short Term ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract Cytokine-based expansion of umbilical cord blood (UCB) in vitro prior to infusion has been pursued in an attempt to overcome the limited cellular content of a single UCB unit. Thus far, these attempts have not shown improvement in kinetics of donor-derived hematopoietic recovery. Our studies have incorporated UCB expanded over a feeder-layer of human mesenchymal stem cells (huMSC), known to inhibit the differentiation of hematopoietic stem cells (HSC) observed in expansion with cytokines alone. Expansion conditions included: UCB expanded over a huMSC monolayer with the addition of cytokines (IL-3, IL-6, G-CSF, SCF, FLT-3L, EPO) and UCB expanded in the same cytokines alone. Day 12 culture readouts included: viable cell counts, 4-color flow analysis, and rates of human engraftment in NOD/SCID mice. In the current study the fold expansion was 6.4 fold in the huMSC + cytokines condition and 7 fold in the cytokines alone condition. Flow cytometry surface marker analysis proportions (absolute numbers) were notable for higher proportions and numbers of early HSC expressing CD133 in cultures incorporating huMSC stromal layer: Unexpanded MSC+ cytokines Cytokines CD34 0.68 (.068M) 0.74 (3.63M) 1.94 (5.39M) CD133 5.69 (.569M) 2.56 (12.54M) 0.74 (2.06M) CD3 49.6 (4.96M) 2.2 (10.78M) 0.42 (1.17M) CD56 17.4 (1.74M) 2.71 (13.28M) 1.06 (2.95M) CD69 0.80 (7.28M) 7.28 (35.67M) 24.4 (67.8M) UCB graft T and NK populations were maintained in huMSC culture conditions and the observed difference in CD69 expression supports the hypothesis that huMSC may have an inhibitory effect on T cell activation during UCB ex vivo expansion. To assess the human engraftment potential of the cultures, cells from each culture condition were injected by tail vein into NOD/SCID mice (no CD34 selection was performed). Mice receiving unexpanded UCB received 10M mononuclear cells each. Mice receiving culture expanded cells received cell doses in proportion to the fold expansion over the number of cells at the initiation of the cultures. Engraftment was assessed by the percentage of human CD45+ (≥0.4%) cells found within the bone marrow of mice at seven weeks post infusion. Mice were injected as follows: 7 mice with unexpanded UCB (2 of which died within a month of transplant), 7 mice with UCB expanded in huMSC + cytokines, and 3 mice with UCB expanded in cytokines alone. Flow analysis of mouse bone marrow cells revealed average CD45+ percentages of 1.79% for mice injected with unexpanded UCB, 2.66% for mice injected with cytokine alone cells, and 5.94% for mice injected with huMSC + cytokine cells. Human cell subset analysis was performed for CD3, CD19, and CD56 content. The percentages of gated CD45+ co-expressing CD3+ were 10.3% in the unexpanded UCB, 16.6% in the cytokine alone condition and 10.4% in the huMSC + cytokine condition. Cells co-expressing CD19+ were 7.86% in the unexpanded UCB, 8.31% in the huMSC + cytokine condition and dropped to 1.43% in the cytokine alone condition. Gated CD45+ cells co-expressing CD56+ were 16.4% in the unexpanded UCB, 8.8% in the huMSC + cytokines condition, and dropped to 2.6% in the cytokines alone condition. In conclusion, UCB expanded short-term in cytokines demonstrates maintenance of earlier HSC phenotype and improved human engraftment in NOD/SCID in cultures incorporating a huMSC monolayer platform.

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