In vitro And In vivo Evidences for the long-term multilineage (Myeloid, b, nk & t) Reconstitution capacity of Ex vivo Expanded 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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Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model

Experimental Hematology ◽

10.1016/j.exphem.2006.04.009 ◽

2006 ◽

Vol 34 (7) ◽

pp. 943-950 ◽

Cited By ~ 27

Author(s):

Yvette van Hensbergen ◽

Laurus F. Schipper ◽

Anneke Brand ◽

Manon C. Slot ◽

Mick Welling ◽

...

Keyword(s):

Mouse Model ◽

Cord Blood ◽

Blood Cells ◽

Scid Mouse ◽

Ex Vivo ◽

Human Cd34 ◽

Scid Mouse Model ◽

Cord Blood Cells ◽

Ex Vivo Culture

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Noninvasive Bioluminescent Imaging Demonstrates Long-Term Multilineage Engraftment of Ex Vivo-Expanded CD34-Selected Umbilical Cord Blood Cells

Stem Cells ◽

10.1002/stem.111 ◽

2009 ◽

Vol 27 (8) ◽

pp. 1932-1940 ◽

Cited By ~ 13

Author(s):

David Steiner ◽

Juri Gelovani ◽

Barbara Savoldo ◽

Simon N. Robinson ◽

William K. Decker ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Blood Cells ◽

Ex Vivo ◽

Bioluminescent Imaging ◽

Cord Blood Cells ◽

Umbilical Cord Blood Cells

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PATTERN OF CYTOKINE EXPRESSION BY RAT LIVER EPITHELIAL CELLS SUPPORTING LONG-TERM CULTURE OF HUMAN CD34+UMBILICAL CORD BLOOD CELLS

Cytokine ◽

10.1006/cyto.1999.0639 ◽

2000 ◽

Vol 12 (7) ◽

pp. 951-959 ◽

Cited By ~ 5

Author(s):

Mickael Rialland ◽

Anne Corlu ◽

Gennady Ilyin ◽

Florian Cabillic ◽

Isabelle Lamy ◽

...

Keyword(s):

Epithelial Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Blood Cells ◽

Human Cd34 ◽

Cord Blood Cells ◽

Umbilical Cord Blood Cells ◽

Rat Liver Epithelial 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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Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.012336799 ◽

2002 ◽

Vol 99 (1) ◽

pp. 413-418 ◽

Cited By ~ 114

Author(s):

C. J. Hogan ◽

E. J. Shpall ◽

G. Keller

Keyword(s):

Cord Blood ◽

Blood Cells ◽

Scid Mice ◽

Human Cd34 ◽

Cord Blood Cells

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Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential

Experimental Hematology ◽

10.1016/s0301-472x(02)00805-6 ◽

2002 ◽

Vol 30 (6) ◽

pp. 612-616 ◽

Cited By ~ 105

Author(s):

Ian K McNiece ◽

Graça Almeida-Porada ◽

Elizabeth J Shpall ◽

Esmail Zanjani

Keyword(s):

Cord Blood ◽

Blood Cells ◽

Ex Vivo ◽

Fetal Sheep ◽

Cord Blood Cells

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Hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood progenitor cells evaluated in vitro and in vivo

Blood ◽

10.1182/blood.v87.4.1261.bloodjournal8741261 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1261-1271 ◽

Cited By ~ 78

Author(s):

DL DiGiusto ◽

R Lee ◽

J Moon ◽

K Moss ◽

T O'Toole ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Cell Cultures ◽

Stromal Cell ◽

Ex Vivo ◽

Hematopoietic Progenitors ◽

Phenotypic Analysis

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)- 3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.

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Expression of TCF3-ZNF384 in Human Hematopoietic Cells Induces Lineage Disruption in Vitro and Acute Leukemia in Vivo

Blood ◽

10.1182/blood-2018-99-118855 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 550-550 ◽

Cited By ~ 1

Author(s):

Kirsten Dickerson ◽

Hiroki Yoshihara ◽

Laura Janke ◽

Charles G. Mullighan

Keyword(s):

Acute Leukemia ◽

Blood Cells ◽

Research Funding ◽

Progenitor Cell ◽

Wild Type ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cord Blood Cells

Abstract Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia. Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice. Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation. Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.

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