Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors

Abstract Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

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Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors

Blood ◽

10.1182/blood.v80.2.403.bloodjournal802403 ◽

1992 ◽

Vol 80 (2) ◽

pp. 403-411

Author(s):

MR Koller ◽

JG Bender ◽

ET Papoutsakis ◽

WM Miller

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Low Oxygen ◽

Human Cord Blood ◽

Cell Production ◽

Endogenous Production ◽

Mature Granulocyte ◽

Marrow Stroma

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

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Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.529 ◽

1993 ◽

Vol 178 (2) ◽

pp. 529-536 ◽

Cited By ~ 89

Author(s):

T Moritz ◽

D C Keller ◽

D A Williams

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Cord Blood ◽

Progenitor Cells ◽

Severe Combined Immunodeficiency ◽

Genetic Material ◽

Retroviral Vectors ◽

Transduction Efficiency ◽

Human Cord Blood

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.

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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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MRI Tracking of FePro Labeled Fresh and Cryopreserved Long Term In Vitro Expanded Human Cord Blood AC133+ Endothelial Progenitor Cells in Rat Glioma

PLoS ONE ◽

10.1371/journal.pone.0037577 ◽

2012 ◽

Vol 7 (5) ◽

pp. e37577 ◽

Cited By ~ 10

Author(s):

Branislava Janic ◽

Kourosh Jafari-Khouzani ◽

Abbas Babajani-Feremi ◽

A. S. M. Iskander ◽

Nadimpalli Ravi S. Varma ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Endothelial Progenitor ◽

Human Cord Blood ◽

Rat Glioma

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Human Cord Blood-Derived AC133+ Progenitor Cells Preserve Endothelial Progenitor Characteristics after Long Term In Vitro Expansion

PLoS ONE ◽

10.1371/journal.pone.0009173 ◽

2010 ◽

Vol 5 (2) ◽

pp. e9173 ◽

Cited By ~ 38

Author(s):

Branislava Janic ◽

Austin M. Guo ◽

A. S. M. Iskander ◽

Nadimpalli Ravi S. Varma ◽

Alfonso G. Scicli ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Endothelial Progenitor ◽

Human Cord Blood ◽

In Vitro Expansion

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Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells

BMC Immunology ◽

10.1186/s12865-017-0209-9 ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 15

Author(s):

Annette Audigé ◽

Mary-Aude Rochat ◽

Duo Li ◽

Sandra Ivic ◽

Audrey Fahrny ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Stem And Progenitor Cells

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The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

Blood ◽

10.1182/blood.2020005357 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2410-2415 ◽

Cited By ~ 1

Author(s):

Peng Hua ◽

Joanna Hester ◽

George Adigbli ◽

Rong Li ◽

Bethan Psaila ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Bet Inhibitor ◽

Functional Studies

Abstract Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

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