Lentivirally Transduced Human Cord Blood CD34+FLT3-ITD+ Cells Induce Murine Acute Leukemia in the NOD/SCID Transplantation Model.

Abstract Abstract 2984 Targeting constitutively activated FLT3 (FLT3-ITD) by tyrosine kinase inhibition (TKI) in acute myeloid leukemia (AML) leads to clearance of blasts in the periphery but not in the bone marrow, suggesting a protective effect of the marrow niche on leukemic stem cells (LSC). We have previously shown that interaction of CD34+FLT3-ITD+ LSC with stromal niche cells mimicking the bone marrow environment specifically protects these cells from the effects of TKI and confers a growth advantage to FLT3-ITD+ leukemic stem/progenitor cells over normal ones (Parmar et al, Cancer Research 2011). To study human FLT3-ITD+ LSC in vivo in the context of the bone marrow niche, we aimed to establish a xenogeneic NOD/SCID mouse model of human FLT3-ITD+AML. Human CD34+ enriched cord blood cells were transduced with a pWPI lentivirus containing a VSV-pseudotyped SIN/LTR vector with eGFP and full length human FLT3 cDNA harboring a 30 bp length internal tandem duplication (FLT3-ITD) or empty vector control. Transduction efficiency ranged between 1–4.4% for FLT3-ITD and 1.3–18% for vector control. Sub-lethally irradiated NOD/SCID mice were then transplanted with 1 × 104 – 7 × 104 unselected or GFP-sorted CD34+FLT3-ITD+ cells. Acute leukemia developed in 7/9 animals after a median latency of 85 days (range 70–168), with involvement of peripheral blood, bone marrow, spleen and liver. Three mice developed acute lymphoblastic leukemia (ALL) whereas the remaining mice showed signs of AML. In contrast, mice receiving empty pWPI vector-transduced human CD34+ cord blood cells (n = 8) all remained healthy during the observation period of 28 weeks and, in 4/8 animals, normal human CD34+cells could be recovered from the bone marrow (human engraftment range 0.3–35.5%). Leukemic mice exhibited hepatomegaly and splenomegaly with an average 10-fold increase in spleen weight, 2-fold increase in spleen length and 2.7-fold increase in liver weight compared to control mice. In mice that developed ALL, lymph node enlargement was also noted. Whole bone marrow, spleen and liver cells from primary mice were re-transplanted and were able to reproduce acute leukemia in all secondary (n=10/10) and tertiary mice (n=11/11) with a median latency of 25 and 20 days, respectively (p<0.01). Surprisingly, detailed immunophenotypical and immunohistochemical analysis revealed all leukemias to be of murine origin. Leukemic cells stained positively for murine CD45.1 antigen but negatively for human CD45. However, a small population of human CD34+CD45+ cells (range 1–7%) was continuously detectable in the bone marrow of primary, secondary and tertiary transplanted leukemic mice. Accordingly, human FLT3-ITD was detectable by PCR specific for human FLT3 up to the third serial transplantation. Viral integration site analysis by LM-PCR on genomic DNA isolated from spleens of leukemic mice revealed lentiviral integration into the human genome, excluding the possibility of in vivo viral shuttling from human cord blood CD34+ cells to mouse hematopoietic stem cells. Moreover, multicolor fluorescent in situ hybridization (M-FISH) on metaphases generated from peripheral blood lymphocytes revealed only murine chromosomes, also ruling out the possibility of fusion between human and mouse cells. To further characterize these murine leukemias, we performed array CGH on murine spleen gDNA of four immunophenotypically different mice. All mice showed recurrent clonal chromosomal aberrations frequently found in AML. Surprisingly, we have no evidence for the presence of FLT3-ITD in these murine leukemias, suggesting that CD34+FLT3-ITD+ stem cells can trigger development of acute leukemia. We propose that leukemogenesis may mechanistically be related to the host microenvironment and that the bone marrow niche in NOD/SCID mice is susceptible to modulation by the FLT3-ITD oncogene. Disclosures: No relevant conflicts of interest to declare.

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High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle

Blood ◽

10.1182/blood-2008-06-161117 ◽

2009 ◽

Vol 113 (12) ◽

pp. 2661-2672 ◽

Cited By ~ 74

Author(s):

Alex J. Tipping ◽

Cristina Pina ◽

Anders Castor ◽

Dengli Hong ◽

Neil P. Rodrigues ◽

...

Keyword(s):

Cell Cycle ◽

Cord Blood ◽

Cell Function ◽

Ki 67 ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cord Blood Cells ◽

Low Efficiency

Abstract Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G0 residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34+CD38−HoechstloPyronin Ylo) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2–conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21CIP1 and p27KIP1 do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2hi) failed to contribute to hematopoiesis in nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2lo cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

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Identification of a New Population of Human Cord Blood Cells with Lymphoid-Restricted Reconstituting Potential in Sublethally Irradiated Immunodeficient Mice.

Blood ◽

10.1182/blood.v104.11.3213.3213 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3213-3213

Author(s):

Oliver Christ ◽

Clayton Smith ◽

Karen Leung ◽

Melisa Hamilton ◽

Connie J. Eaves

Keyword(s):

Cord Blood ◽

Blood Cells ◽

Adult Life ◽

Lymphoid Cells ◽

Human Cord Blood ◽

Immunodeficient Mice ◽

Post Transplant ◽

Cord Blood Cells

Abstract Throughout adult life, human hematopoiesis is sustained by the activity of a small compartment of pluripotent stem cells with extensive self-renewal potential. Available evidence suggests that these cells undergo a process of progressive lineage restriction similar to that described for murine hematopoietic cells, although many of the intermediate stages of human hematopoiesis have not yet been characterized. In human hematopoietic tissues, cells with short-term (<4 months) as well as long term (>4 months) repopulating activity (termed STRCs and LTRCs, respectively) are distinguished by their differential ability to engraft sublethally irradiated NOD/SCID-β2microglobulin null mice as well as their transient versus sustained output of differentiated cells. In previous studies, both a myeloid-restricted type of human STRC (STRC-M) and a type of STRC with lymphoid as well as myeloid potential (STRC-ML) have been identified. STRC-Ms are CD34+CD38+ and produce mainly erythroid progeny for the first 3–4 weeks post-transplant. In contrast, STRC-MLs are CD34+CD38− and produce progeny only between weeks 5 and 12 post-transplant which consist mainly of B-lymphoid cells plus some granulopoietic cells. We show here that both STRC-MLs and STRC-Ms are similarly distributed among lin- cord blood cells with intermediate to high levels of aldehyde dehydrogenase activity (ALDH-int/hi) as evidenced by staining with the fluorescent dye BAAA. In addition, BAAA-staining has allowed a previously undescribed primitive cell with low ALDH activity (ALDH-lo) and lymphoid-restricted repopulating activity to be identified. Assessment of NOD/SCID-β2microglobulin null mice transplanted with various subsets of cord blood cells further demonstrated that these “STRC-Ls” are CD38− and 10-fold more prevalent in the CD133+ subset of the low-density SSC-low ALDH-lo/neg population but, numerically, are equally distributed between the CD133+ and CD133− fractions because of the proportionately larger size of the CD133− subpopulation. Phenotype analysis of CD34+CD38− cord blood cells revealed a small and distinct ALDH-lo subset that expressed 10-fold higher levels of CD7 than any other CD34+CD38− cells. However, transplantation of this small CD7++ subset into NOD/SCID- β2microglobulin null mice revealed that they accounted for very few of the ALDH-lo STRC-Ls. The discovery of a CD38− ALDH-lo population of lymphoid-restricted human cells with in vivo reconstituting activity identifies a key step in the process of human hematopoietic cell lineage determination and the ability to prospectively isolate these progenitors separately from other types of short- and long-term repopulating cells present in normal human hematopoietic tissues should greatly facilitate future analysis of the mechanisms regulating their normal differentiation or malignant transformation.

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Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽

10.1182/blood.v108.11.3650.3650 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3650-3650

Author(s):

Kent W. Christopherson ◽

Tiki Bakhshi ◽

Shamanique Bodie ◽

Shannon Kidd ◽

Ryan Zabriskie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Blood Cells ◽

Hematopoietic Stem ◽

Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

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Time Course Studies of the Progeny of Barcoded CD34+ Human Cord Blood Cells Generated in Transplanted NOD/SCID-IL2Rγ−/− Mice Unveil the Clonal Dynamics of Short Term, Intermediate Term and Long Term Repopulating Cells

Blood ◽

10.1182/blood.v120.21.28.28 ◽

2012 ◽

Vol 120 (21) ◽

pp. 28-28

Author(s):

Alice M.S. Cheung ◽

Long V. Nguyen ◽

Annaick Carles ◽

Paul H. Miller ◽

Philip A Beer ◽

...

Keyword(s):

Cord Blood ◽

B Cell ◽

Blood Cells ◽

Bone Marrow Cells ◽

Self Renewal ◽

Post Transplant ◽

Human Cd34 ◽

Cord Blood Cells

Abstract Abstract 28 Hematopoietic stem cells (HSC) exhibit heterogeneity in self-renewal and differentiation activity, but the extent to which this is intrinsically determined and extrinsically regulated is still poorly understood. In the mouse, purities of HSCs can now be achieved to allow such questions to be addressed directly. Interestingly, tracking the outputs of large numbers of serial transplantable clones produced from single-cell transplants, or the clonal progenies of vector-marked/barcoded cells indicate the existence in mice of 2 subsets of HSCs with durable self-renewal ability. These 2 subsets are characterized by distinct lineage output programs that are maintained through the many HSC self-renewal divisions required to serially propagate a clone in vivo. To begin to ask whether similar subsets of human HSCs exist, we have created a diverse lentiviral library encoding an estimated >105 different barcode sequences and GFP, and then used this library to track the in vivo clonal outputs of transduced human CD34+ cord blood cells in xenografted mice. For this experiment, CD34+ cells isolated immunomagnetically to a purity of >80% were exposed to virus for 6 hours in the presence of growth factors and then immediately injected intravenously into 2 sublethally irradiated NOD/SCID-IL2Rγ−/− mice (1.2 × 105 cells per mouse; 30% GFP+ cells after 3 days in vitro). Different subsets of human cells were then isolated by FACS from immunostained bone marrow cells aspirated sequentially from the femurs of the mice at intervals from 4–27 weeks post-transplant and the identity, number and size of clones in each established by next generation sequencing of barcoded amplicons derived from each sample. To identify barcodes arising from PCR and sequencing errors and calibrate clone sizes, we included 3 controls of 20, 100 and 500 cells with a known barcode at each datapoint. The data from these controls allowed a threshold of 20 cells per clone to be established with >95% confidence. We then compared the representation of clones among all samples from each mouse to derive the number and size of all clones detected, assuming a mouse contains 2×108 bone marrow cells. This analysis revealed a total of 154 uniquely barcoded clones containing up to 2×108 human hematopoietic cells in the 2 mice (8–30 × 106 in one and 4–165 × 106 in the other at any single time point). Analysis of the representation of each clone over time showed successive waves of repopulation from different clones with lineage output profiles consistent with those obtained by transplanting separate fractions of CD34+ cord blood cells distinguished by their surface phenotypes. Specifically, we detected 50 clones (32% of all clones) that were not sustained at detectable levels beyond 9 weeks post-transplantation and were characterized by robust myeloid differentiation with variable B cell outputs at 4 weeks. Another 30 clones (19%) showed significant but also transient outputs of either or both the myeloid and B cell lineage, disappearing between week 9 and 16 post-transplant. Mature cell output was detected from a total of 74 clones (48%) at the 27 week time point, among which 36 (23%) were not evident during the first 4 months post-transplant. These late-appearing clones were mostly small (contributing up to 3 × 105 total hematopoietic cells at week 27) and made a significantly higher contribution to the total human myeloid population than to the total human B cell population. Notably, the 12 long term clones that showed robust mature cell output detectable in all 3 sites sampled at week 27 when the mice were sacrificed (left leg vs right leg vs pelvis) contained both myeloid and lymphoid cells but with large (>100-fold) variations in their representation in the 3 different sites. This latter finding suggests less trafficking of human cells between sites than expected from parabiotic mouse experiments or substantial differences in the differentiation control exerted in different locations. Additionally, from one of the mice, we obtained the first direct evidence of a large output of human T cells (>9 × 106) that was part of a long term multi-lineage clone detectable at 27 weeks post-transplant. This first use of a barcoding strategy to analyze the clonal dynamics of normal human CD34+ cells with in vivo repopulating activity demonstrates the power of this approach to analyze their lineage outputs and sets the stage for novel applications to expanded and transformed populations. Disclosures: No relevant conflicts of interest to declare.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Identification of Lymphomyeloid Primitive Progenitor Cells in Fresh Human Cord Blood and in the Marrow of Nonobese Diabetic–Severe Combined Immunodeficient (NOD-SCID) Mice Transplanted with Human CD34+ Cord Blood Cells

Journal of Experimental Medicine ◽

10.1084/jem.189.10.1601 ◽

1999 ◽

Vol 189 (10) ◽

pp. 1601-1610 ◽

Cited By ~ 60

Author(s):

Catherine Robin ◽

Françoise Pflumio ◽

William Vainchenker ◽

Laure Coulombel

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Scid Mice ◽

Human Cord Blood ◽

Human Cd34 ◽

Nonobese Diabetic ◽

Direct Demonstration ◽

Cd34 Cells ◽

Cell Assays ◽

Cord Blood Cells

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34+ cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34+ CD19−Thy1+ (or CD38−) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19+, CD56+, and CD15+ cells in stromal cocultures and CD4+CD8+ T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34+ cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34+ cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

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Expression of P16 cell cycle inhibitor in human cord blood CD34+ expanded cells following co-culture with bone marrow-derived mesenchymal stem cells

Hematology ◽

10.1179/1607845412y.0000000009 ◽

2012 ◽

Vol 17 (6) ◽

pp. 334-340 ◽

Cited By ~ 7

Author(s):

Arezoo Oodi ◽

Mehrdad Noruzinia ◽

Mehryar Habibi Roudkenar ◽

Mahin Nikougoftar ◽

Mohamad Soleiman Soltanpour ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Human Cord Blood ◽

Cell Cycle Inhibitor ◽

Cord Blood Cd34

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Identification of novel circulating human embryonic blood stem cells

Blood ◽

10.1182/blood.v96.5.1740 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1740-1747 ◽

Cited By ~ 33

Author(s):

Lisa Gallacher ◽

Barbara Murdoch ◽

Dongmei Wu ◽

Francis Karanu ◽

Fraser Fellows ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cellular Therapy ◽

Fetal Liver ◽

In Utero ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Fetal Stem Cells

Abstract Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 × 105, illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized–peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies.

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

Blood ◽

10.1182/blood.v108.11.3184.3184 ◽

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.

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Umbilical Cord Blood CD133+ Stem Cells Exhibit Vasculogenic Capacity In Vitro and Augment Neovasculogenesis In Vivo in a Murine Model of Vascular Ischemia.

Blood ◽

10.1182/blood.v108.11.1790.1790 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1790-1790

Author(s):

M.R. Finney ◽

L.R. Fanning ◽

P.J. Vincent ◽

D.G. Winter ◽

M.A. Hoffman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Blood Flow ◽

Cord Blood ◽

Umbilical Cord Blood ◽

Hind Limb ◽

Capillary Density ◽

Functional Assays

Abstract Recent reports have utilized a variety of cell types for cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to assess the vasculogeneic potential of selected CD133+ hematopoietic stem cells (HSC) from umbilical cord blood (UCB) utilizing in vitro functional assays and an in vivo murine hind-limb ischemia model. Methods & Results: Mononuclear cells (MNC) from UCB or bone marrow (BM) were incubated with CD133+ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133+ cells was 0.5±0.2% of UCB MNC and 0.7±0.3% of BM MNC, with a purity of 79±2% (UCB, n=30) and 84±5% (BM, n=12). Surface expression in the UCB CD133+ population was 3.6±1.5% KDR(VEGFR2), 8.7± 3.8% CXCR4 and 22.7±2.8% CD105 compared to 9.2±1.8% KDR, 14.4±1.3% CXCR4 and 23.7±2.3% CD105 in the BM CD133+ population. We measured chemotactic migration of cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. The fold increase over control was 4.9±2.9 UCB MNC, 1.8±0.7 UCB CD133+ and 8.3±1.7 BM CD133+ (n=3). Angiogenic protein assays of CD133+ cells demonstrated elevated levels of IL-8 production as compared to MNC (103+/−380 pg/mL greater in CD133+ than MNC from the same UCB unit) when cultured for 24h in basal media. NOD/SCID mice underwent ligation of the right femoral artery and were given cells or vehicle control via intracardiac injection immediately following injury. Mice were given 1 x 106 MNC or 0.5 x 106 CD133+ cells. Laser Doppler flow measurements were obtained from both limbs each week for 6 weeks and the ratio of perfusion in the ischemic/healthy limb was calculated. At 28 days, perfusion ratios were statistically higher in study groups receiving UCB CD133+ cells, 0.55±0.06 (n=9), BM CD133+ cells 0.47±0.07 (n=8), BM MNC 0.48±0.8 (n=6) compared to cytokine controls 0.37±0.03 (n=12, p<0.05). Mice receiving UCB MNC did not show statistically significant improvement in measured blood flow over control animals 0.42±0.05 (n=8, p=0.34). At sacrifice, bone marrow was harvested to assess engraftment of human cells by flow cytometric analysis. Mice injected with UCB CD133+ cells showed 19±4.9% positive huCD45 cells compared to 2.5±0.6% for UCB MNC, 1.6±0.4% for BM CD133+ cells and 2.3±0.3% for BM MNC (n=3). Histological studies from day 42 tissue samples of muscle distal to arterial ligation were evaluated for capillary density. Control animals had capillary density of 131±6.9 cells/mm2. Capillary density was statistically higher that controls in animals receiving UCB CD133+ (320±18; p<0.0001), BM CD133++ (183±9.3; p<0.0001), and UCB MNC (164±10.5; p=0.011). Mice treated with BM MNC (135±9.4) did not have a statistically significant increase in capillary density from controls (p=0.73). In addition, animals treated with either UCB or BM-derived CD133+ cells had statistically higher capillary density than unselected MNC (p=<0.0001 and p=0.0004, respectively). Conclusions: In vitro functional assays showed that UCB-derived CD133+ HSC demonstrate enhanced homing capability (migration) as well as the potential for cellular recruitment (via IL-8 production) for angiogenesis in response to ischemia. Furthermore, UCB derived CD133+ HSC mediate significantly improved blood flow in an in vivo murine hind-limb injury model of ischemia, indicating the greater vasculogenic potential of selected CD133+ cells from of this stem cell source.

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