Flt3 Ligand Enhances the Yield of Primitive Cells After Ex Vivo Cultivation of CD34+ CD38dim Cells and CD34+ CD38dim CD33dim HLA-DR+ Cells

Abstract Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma-free culture system. Towards that end, four CD34+ subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit–granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- ± 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33dim HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim cells and CD34+ HLA-DRdim cells after 5 to 12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34+ CD38dim or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P < .05). For HLA-DR+ cells, LTCIC recoveries averaged 214% ± 87% of input with FL and 24% ± 16% without FL. In contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34+ cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34+ cells, and CFU-GM and enhances the pool of early CD34+ CD33dim cells, CD34+ HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool.

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Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v128.22.1471.1471 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1471-1471

Author(s):

Munetada Haruyama ◽

Kozo Yamaichi ◽

Akira Niwa ◽

Megumu K Saito ◽

Tatsutoshi Nakahata

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Absolute Number ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Mean

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet. To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis. The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs. We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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Expression of CD4 by human hematopoietic progenitors [see comments]

Blood ◽

10.1182/blood.v84.10.3344.3344 ◽

1994 ◽

Vol 84 (10) ◽

pp. 3344-3355 ◽

Cited By ~ 67

Author(s):

F Louache ◽

N Debili ◽

A Marandin ◽

L Coulombel ◽

W Vainchenker

Keyword(s):

Functional Analysis ◽

Large Fraction ◽

Hematopoietic Progenitors ◽

Differentiation Markers ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Colony Forming Unit ◽

Hla Dr ◽

Cd34 Cells

Abstract It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.

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Expression of CD4 by human hematopoietic progenitors [see comments]

Blood ◽

10.1182/blood.v84.10.3344.bloodjournal84103344 ◽

1994 ◽

Vol 84 (10) ◽

pp. 3344-3355 ◽

Cited By ~ 1

Author(s):

F Louache ◽

N Debili ◽

A Marandin ◽

L Coulombel ◽

W Vainchenker

Keyword(s):

Functional Analysis ◽

Large Fraction ◽

Hematopoietic Progenitors ◽

Differentiation Markers ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Colony Forming Unit ◽

Hla Dr ◽

Cd34 Cells

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.

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Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells.

Journal of Experimental Medicine ◽

10.1084/jem.184.4.1357 ◽

1996 ◽

Vol 184 (4) ◽

pp. 1357-1364 ◽

Cited By ~ 97

Author(s):

S Tajima ◽

K Tsuji ◽

Y Ebihara ◽

X Sui ◽

R Tanaka ◽

...

Keyword(s):

Interleukin 6 ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Human Cd34 ◽

Novel Approach ◽

Cd34 Cells ◽

Interleukin 6 Receptor ◽

Serum Free Suspension

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

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Phenotypic and functional characterization of long-term culture- initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor

Blood ◽

10.1182/blood.v88.6.2033.bloodjournal8862033 ◽

1996 ◽

Vol 88 (6) ◽

pp. 2033-2042 ◽

Cited By ~ 57

Author(s):

F Prosper ◽

D Stroncek ◽

CM Verfaillie

Keyword(s):

Steady State ◽

Absolute Number ◽

Granulocyte Colony Stimulating Factor ◽

Hla Dr ◽

Normal Individuals ◽

Cd34 Cells ◽

The Absolute ◽

Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) have successfully been used as stem cells for both autologous and allogeneic transplants. However, little is known concerning the absolute number and phenotype of primitive progenitors, such as long-term culture-initiating cells (LTC-IC) in mobilized PBPC. The aim of our study was to evaluate the capacity of G- CSF to mobilize LTC-IC in the PB of normal individuals and to evaluate the phenotypic and functional characteristics of G-CSF mobilized LTC- IC. G-CSF was administered to 29 healthy volunteers at 7.5 micrograms or 10 micrograms/kg/d subcutaneously (SC) for 5 consecutive days and PBPC were harvested on day 6. Mobilization with G-CSF increased the absolute number of week 5 LTC-IC in PB 60-fold, while the number of CD34+ cells and committed colony forming cells (CFC) was increased sevenfold to 12-fold. The frequency of CFC and week 5 LTC-IC in CD34+ cells selected by fluorescence-activated cell sorter (FACS) from mobilized PBPC was 2 +/- 0.3-fold and 9 +/- 2.2-fold higher respectively than in CD34+ cells selected from unmobilized PBMNC. CFC were enriched in the CD34+ CD38+ and CD34+ HLA-DR+ populations. The absolute number of LTC-IC present in CD34+ CD38- and CD34+ HLA-DR- cells selected by FACS from either mobilized PBPC, unmobilized PBMNC or steady state bone marrow (BM) was similar (0.5% to 2%). In contrast to unmobilized PBMNC or steady state BM CD34+ CD38+ and CD34+ HLA-DR+ cells, which contain less than 0.1% LTC-IC, CD34+ CD38+ and CD34+ HLA- DR+ cells sorted from mobilized PBPC contained 0.5% to 5% of cells capable of sustaining hematopoiesis in long-term cultures for 5 weeks. However, 90% to 95% of LTC-IC present in mobilized CD34+ CD38+ and CD34+ HLA-DR+ cells were not able to sustain hematopoiesis for 8 weeks, while 30% of CD34+ CD38- and CD34+ HLA-DR- LTC-IC present in mobilized PBPC could sustain hematopoiesis for at least 8 weeks. This suggests that the majority of CD34+ CD38+ and CD34+ HLA-DR+ week 5 LTC-IC represent progenitors at an intermediate state of differentiation. We conclude that G-CSF effectively mobilizes LTC-IC in the blood of normal individuals. Although a fraction of these cells has functional characteristics similar to those of steady state PBMNC or BM LTC-IC, more than 85% of mobilized PBPC LTC-IC are CD34+ CD38+ and CD34+ HLA- DR+, capable of sustaining hematopoiesis for 5 weeks, but not for 8 weeks. The functional and phenotypic characterization of primitive and more mature populations of LTC-IC in mobilized PBPC should prove extremely useful in future studies examining the role of these progenitors in engraftment following transplantation.

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Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Blood ◽

10.1182/blood.v130.suppl_1.709.709 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 709-709

Author(s):

Bin Guo ◽

Xinxin Huang ◽

Hal E. Broxmeyer

Keyword(s):

Ex Vivo ◽

Ex Vivo Expansion ◽

Metabolic Reprogramming ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Ppar Γ ◽

Control Shrna ◽

Cd34 Cells ◽

Human Hscs

Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.

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Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽

10.1182/blood.v91.4.1243 ◽

1998 ◽

Vol 91 (4) ◽

pp. 1243-1255 ◽

Cited By ~ 69

Author(s):

Mo A. Dao ◽

Ami J. Shah ◽

Gay M. Crooks ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Low Levels ◽

Ex Vivo Culture ◽

Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

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Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽

10.1182/blood.v92.12.4641 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4641-4651 ◽

Cited By ~ 105

Author(s):

Pankaj Gupta ◽

Theodore R. Oegema ◽

Joseph J. Brazil ◽

Arkadiusz Z. Dudek ◽

Arne Slungaard ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Stem Cell Niche ◽

Ex Vivo ◽

Recent Observation ◽

Hematopoietic Progenitors ◽

Platelet Factor ◽

Cell Niche

Abstract Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

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Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Blood ◽

10.1182/blood.v104.11.4954.4954 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4954-4954

Author(s):

Ronald L. Brown ◽

J. Zhang ◽

L. Qiu ◽

A. Nett ◽

G. Almeida-Porada ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Culture Conditions ◽

Cell Populations ◽

Multilineage Differentiation ◽

Cd34 Cells ◽

Ex Vivo Culture ◽

Cytokine Combination ◽

Cells Cultured

Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.

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High Efficiency Recovery of Hematopoietic Progenitors from Peripheral Blood Harvests after Short- and Long-Term Cryopreservation.

Blood ◽

10.1182/blood.v106.11.5275.5275 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5275-5275

Author(s):

Ulrich Denz ◽

Dagmar Wider ◽

Antonia Mueller ◽

Monika Engelhardt

Keyword(s):

Suspension Culture ◽

Peripheral Blood ◽

High Efficiency ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Viable Cells ◽

Cd34 Cells ◽

Significant Difference ◽

Group A

Abstract Introduction: Transplantation of functional hematopoietic stem cells (HSC) using peripheral blood (PB), bone marrow (BM) or cord blood (CB) cells is widely used to treat malignant and nonmalignant disorders. Because long-term cryopreservation is performed for PB, BM and CB cells, and these are often used years after cell harvests, the implementation of a quality-assurance is a major requirement to ensure graft safety for clinical use. Methods: We assessed the efficiency of recovery of viable HSC from 37 patients (pts; n=20 NHL, n=6 Hodgkin, n=9 MM, n=2 AML) and 6 allogeneic-donors (AD) with stored PBSC samples. All pts had received an auto-PBSCT between 1992–2004. Stored PBSC samples used in this analysis had been cryopreserved for a median of 5.6 years (y; range: 1.3–12). We determined post-thawing recovery, cell viability, ex vivo expansion potential, CD34+ numbers, CFU growth in methylcellulose culture and LTC-ICs. Viable cells were determined by trypan blue and propidium iodide via FACS analysis, CFUs in 0.9% methylcellulose (supplemented with IMDM, 30% FCS and EPO, IL-3+GM-CSF) and LTC-IC as previously described. Pts and AD were analyzed as a total group and within 3 subgroups of: A) ‘long-term’ cryopreservation: n=21 PBSC harvests had a median cryopreservation of 9.5y (8–12), B) ‘short-term’ cryopreservation: n=16 harvests had a 2.9y (1.3–5.6) cryopreservation period, and C) n=6 pts showing delayed engraftment (EG) or early death after auto-PBSCT: the cryopreservation in these 6 pts was 2.7y (2.2–3.5). Cryopreservation results were correlated with clinical results and EG. Results: Hematopoietic EG in group A and B was prompt with WBC>1000/μl and platelets>20,000/μl on d10–11 post PBSC reinfusion. EG in group C was delayed albeit 4.3x106 CD34+ cells/kg bw (2.1–8.6) had been retransfused (WBC>1000/μl + platelets>20,000/μl: d+13 post PBSC infusion, non-platelet-EG >20,000/μl before death: n=5). Primary cause of death in group C was progressive disease in 3 and serious infections in 5 pts. Group A showed 74.3% viable cells post-thawing in PBSC grafts. Median number of CD34+ cells were 2.9%. Median numbers of CFU-C, BFU-E and GEMM were 36, 60 and 7, respectively. This was comparable with results in group B, showing 70% viable cells post-thawing, CD34+ cells of 4.2% and CFUs of 43, 75 and 6, respectively (p>0.05). Proliferative capacity was intact in both groups after 7 days of suspension culture, generating CFU-C, BFU-E and GEMM of 67, 29 and 1, respectively. In group C, viable cells were present in only 58% and median CFU-C, BFU-E and GEMM were 21, 5 and 0, respectively (p<0.05). After 7 days of suspension culture, total CFUs were 5 (<5% as compared to group A+B). Mean CFU-Cs before and after LTC-IC were 9 and 8 after LTC-IC culture in group C, whereas these were 18 and 16 in group A (p<0.05). Thus, the percentage of viable cells, CFUs and LTC-ICs was preserved after long-term cryopreservation (group A), showed no significant difference between group A+B, but were decreased in group C. Conclusions: We show that human PBSC can be stored for more than a decade without apparent loss of HSC activity and can be efficiently retrieved. These results reinforce that expiration dates cannot be set for safely stored cryopreserved HSC. Assessment of CD34+ cell numbers, clonogenic potential via methylcellulose and LTC-IC assays are clinically relevant, since they may correlate with clinical outcome. Thus, these hematopoietic assays are valuable to assess the quality of cryopreservation and possibly also outcome of PBSCT.

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