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Keywords:

  • cord blood cells;
  • ex vivo expansion;
  • growth factors;
  • haematopoietic stem cells;
  • megakaryocytopoiesis

Summary

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34+ HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.

Severe prolonged thrombocytopenia is a life threatening complication encountered in cancer patients treated with high-dose chemotherapy followed by transplantation of haematopoietic stem/precursor cells (HSPC) and is most pronounced following cord blood transplantation (CBT) (Brunstein et al, 2007a,b). While umbilical cord blood (CB) provides an important source of HSPC for allogeneic transplantation in young patients, its use in adults is limited by the inadequate number of primitive stem cells and megakaryocyte progenitors (Mk-p) in single or even double CB units (Kanamaru et al, 2000; Ballen et al, 2007), resulting in delayed engraftment until the long-term engrafting cells can support platelet production. The only current treatment for these patients, to reduce their risk of severe bleeding, remains costly multiple platelet transfusions that carry blood products risks, with human leucocyte antigen (HLA) alloimmunization occurring in up to 30% of individuals (Kaushansky, 2008). Administration of thrombopoietin (TPO) or TPO receptor agonists is not clinically effective following severe myeloablation or stem cell transplantation due to low number of cytokine responsive progenitors in the grafts (Evangelista et al, 2007).

A new therapeutic strategy could involve transplantation of ex vivo-generated Mk-p together with unmanipulated single or double CB units. Ex vivo expanded Mk were administered to patients receiving autologous peripheral blood transplant with no recorded toxicity (Bertolini et al, 1997). Pre-incubation of bone marrow (BM) cells with interleukin 3 (IL-3)+ granulocyte-macrophage colony-stimulating factor (GM-CSF) shortened platelet recovery following BM transplantation (Naparstek et al, 1992) and stimulated ex-vivo expansion of Mk-p (Nagler et al, 1995). Improved Mk-p engraftment was reported in mice that received CB CD34+ cells cultured for 4 weeks with cytokine combinations and thrombopoietin (TPO) (Bruno et al, 2004). However, expanding purified CD34+ HSPC is not practical for CBT due to the limited number of indispensable stem cells in the CB units and the long culture period required for effective expansion (De Bruyn et al, 2005). Moreover, these cultures produced mature blood cells rather than immature HSPC, with no reliable long-term bone marrow reconstitution (Hofmeister et al, 2007) and clinical trials with short term expanded CD34+ cells did not improve platelet engraftment (McNiece et al, 2000; Shpall et al, 2002). For these reasons we chose to expand haematopoietic precursors from a small aliquot of whole CB under conditions that partially mimicked the human BM niche, to promote survival and proliferation of HPSC and Mk-p in the mononuclear cell (MNC) fraction. This was performed by coating the culture platform with fibronectin (FN) and supplementing the cultures with factors known to drive expansion of both severe combined immunodeficiency (SCID) repopulating HSPC and Mk (Fox et al, 2002; Feng et al, 2006; Pick et al, 2006; Sagar et al, 2006; Yeoh et al, 2006). We considered FN to be a prime candidate to support survival and proliferation of HSPC and Mk-p in vitro because FN is a major extracellular matrix (ECM) component of all haematopoietic niches, expressed abundantly on the surface of mesenchymal and stromal cells, which are adjacent to HSPC and Mk-p in the BM, and it enhances viability and proliferation of HSPC (Avecilla et al, 2004; Hitchcock & Kaushansky, 2007). Other growth stimulators added were TPO, the major physiological stimulator of Mk (Kaushansky, 2005; Deutsch & Tomer, 2006) which drives all stages of megakaryocytopoiesis and thrombopoiesis, the stress-associated acetylcholinesterase readthrough, peptide (ARP), known to stimulate proliferation of HSPC and Mk maturation (Grisaru et al, 2001, 2006; Deutsch et al, 2002; Pick et al, 2006)and β-fibroblast growth factor (β-FGF) (Yeoh et al, 2006).

We report for the first time impressive simultaneous expansion of both HSPC and Mk-p and Mk maturation. Additionally, we described the subpopulations of Mk-p and HSPC that were expanded, which included the haematopoietic progenitors (CD34high Side Scatter SSClow) and their highly resolved CD41high or CD41low and/or CD33 populations subpopulations, and Mk-p (SSClow CD45dim/neg, CD41high) subsets expressing CD34 or CD33 that contributed to the overall progenitor population. These highly proliferative early HSPC and Mk-p populations, which are capable of maturation, may now provide the cells needed for long term engraftment and for immediate thrombopoiesis during the platelet nadir post-transplant period, with minimal sacrifice of the stem cells in the CB unit. This approach may lead to the development of more practical and effective cell therapy modalities to facilitate myelopoiesis and thrombopoiesis and shorten protracted thrombocytopenia following transplantation.

Material and methods

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

Cell preparation

This study was approved by the hospital ethics committee according to the Helsinki Accords. Samples of 20–40 ml CB were collected from placentas intended for disposal after normal deliveries, following signed informed consent from mothers (N = 20), in heparinized tubes and processed within 8 h. The MNC fraction was isolated on gelatin followed by ficoll-paque gradient to minimize the loss of progenitors (Pick et al, 1998) as detailed in Appendix S1 and Table SI.

Progenitor expansion

The MNC were cultured (0·5 × 106/ml) in tissue culture flasks coated or uncoated with human FN (50 μg/ml; Sigma Chemical Co, St Louis, MO,USA) with the following growth supplements: recombinant human (r-hu)-TPO (10 ng/ml) and Stem cell factor (r-hu-SCF; R&D, Minneapolis, MN, USA), β–FGF (10 ng/ml; kindly provided by Prof I Vlodavsky from the Rappaport Faculty of Medicine, Technion, Haifa, Israel), or synthetic ARP (2 nmol/l), a newly discovered potent haematopoietic growth stimulating peptide derived from the stress variant of acetylcholinesterase (Deutsch et al, 2002; Grisaru et al, 2006; Pick et al, 2006) kindly provided by Prof H Soreq, the Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University, Jerusalem, and supplemented with 10% autologous plasma. Growth factors were supplemented every 3 days. Cell counts were determined microscopically using a hemocytometer or with a Coulter Counter LH500 automated analyser (Beckman-Coulter Inc, Miami, FL, USA) and viability monitored by fluorescent propidium iodide (PI) stain or by trypan blue dye exclusion. The 8–10 d cultured progenitor subpopulations were compared to the initial populations at day 0.

Identification of HSPC and megakaryocyte progenitors and their subpopulations by high resolution flow cytometry

A stringent gating scheme was developed to identify true HSPC and Mk-p and to eliminate non-relevant cells from the analysis (Appendix S1). The distinct blood cell populations were detected by four colour flow cytometry (FC), staining cells with saturating amounts of fluorescently-labelled monoclonal antibodies or isotype antibodies conjugated to the identical fluorochromes as controls and setting appropriate sequential gates to enable high resolution FC analysis of Mk, Mk-p and HSPC. HSPC were assessed using peridinin chlorophyll (perCP)-CD45, phycoerythrin (PE)-CD34 (BD Bioscience, San Jose, CA, USA), monocytes and myeloid cells with allophycocyanin (APC)-CD33 or APC-CD14 and perCP-CD45 and Mk with perCP-CD45 and fluorescein isothiocyanate (FITC)-CD41 [glycoprotein (GP) IIb] (Beckman Coulter). FC analysis was performed using a FACS Calibur (BD Bioscience, Franklin Lakes, NJ, USA) equipped with two power 15 mW argon lasers. Data analysis and significance were calculated using BD Diva 6.1.1 or Cell Quest Pro software (BD Bioscience). Total cell gate included lymphocytes, monocytes and granulocytes with least 100 000 events analysed for each sample. Cultured cells underwent Fc-receptor blocking with goat anti-human IgG to reduce nonspecific staining. HSCP were identified as SSC (side scatter)med/low/CD34high cells. Nonspecific staining of HSPC was determined by staining cells with an isotype control PE or FITC conjugates (Becton-Dickinson). Mk-p identification required a stepwise high definition gating scheme to eliminate unwanted cells and clearly resolve the expanded progenitors and their subpopulations. The first gate eliminated dead cells and debris in the cultures. A second gate was set to include only the live CD45dim/neg/SSCmed/low progenitor cells. This step removed the CD45high and SSChigh cells from the analysis, eliminating the non-HSPC and non-Mk haematopoietic cells that underwent proliferation and excluded mature haematopoietic cells that acquired CD41 labelling in culture (see Appendix S1). As Mk do not express significant levels of CD45, Mk-p were identified as CD45dim/neg/SSCmed/low/CD41high. Subpopulations within CD34high HSPC and CD45dim/neg/SSCmed/low/CD41high Mk-p were further identified.

Haematopoietic colony forming progenitors and their identification

Fresh CB MNC or MNC after 10 d in culture under different conditions were assayed using 2 × 105 cells/well for progenitor-derived colony forming units. Colonies were grown in Methocult GF H4434 containing cytokines (Stem Cell Technolgies Inc, Vancouver, BC, Canada). Mk colonies were grown in Methocult GF H4434 or plasma clots supplemented with TPO. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Mk colony forming units (CFU-Mk) were identified microscopically and by indirect immunofluorescence as clusters of three or more Mk on day 12 (Deutsch et al, 1995). The multilineage colonies (CFU-GEMM) of more than 100 cells containing cells of the granulocyte, monocyte, erythroid and Mk lineages and granulocyte-macrophage CFU (CFU-GM) were counted on day 14 (Grisaru et al, 2001).

Megakaryocyte maturation by DNA ploidy analysis

The measurement of DNA content and of surface expression of CD41were used to investigate ploidy of Mk cells. The cells were labelled with anti-CD41-FITC. The surface stained cells were washed in phosphate-buffered saline before PI labelling by adding permeabilizing reagent and vigorous mixing. PI solution (DNA prep stain; Beckman Coulter) was then added, and the cells incubated for 24 h at 4°C in the dark prior to DNA analysis by FC.

Molecular quantification using real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted with an RNAqueous 4 PCR kit (Ambion; Applied Biosystems, Carlsbad, CA, USA). c-DNA was generated by RT-PCR (FRG; Boehringer Mannheim, Mannheim, Germany) using mRNA (0·5 μg) reverse transcribed with oligo dT primers. Quantitative expression of ITGA2B (GPIIb; CD41) mRNA was performed by RT-PCR with precalibrated and validated primer sets (Rosenberg et al, 2003) using the LightCycler technology (Roche, Basel, Switzerland). A standard curve was generated using the Meg01 cell line c-DNA. RT-PCR expression of ITGA2B was performed using 300 000 non-adherent nucleated cells from fresh CB or after 10 d in culture grown under the above specified conditions. Each c-DNA sample was analysed for ITGA2B (the target gene) and for GAPDH (a housekeeping gene). The results were normalized in each sample to GAPDH expression to eliminate processing errors.

Statistical analysis

Results are expressed as mean ± standard deviation (SD). Statistical significance was determined using the Student t-test and analysis of variance (anova). P-values ≤0·05 were considered significant.

Results

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

Enhancement of viability and ex-vivo expansion of HPSC and Mk-p by FN

Fibronectin is a principal adhesive protein of the BM hematopoietic niche, known to support the survival and proliferation of HSPC. Progenitor-enriched CB MNC were therefore grown in FN-coated flasks supplemented with new combinations of growth modulators present in the BM during haematopoietic stress and known to drive SCID repopulating cells. These included r-hu-TPO, r-hu-SCF, β-FGF and ARP. As expected, cells grown on FN alone, or in combination with components of the hematopoietic niche, displayed increased viability and proliferation at the end of the culture period (Table I).

Table I.   Fibronectin protects cord blood MNC from death.
 ControlARPTPOFNFN + TPOFN + ARPFN + TPO + ARP
  1. Cord blood MNC (105) were cultured for 8–10 d with or without the indicated growth stimulators. Data represent the average percentage of live or dead cells ± SD and cell numbers ± SD from at least five individual cultures. At the termination of cultures cell death was analysed by flow cytometry using propidium iodide staining, viability was assessed by trypan blue dye exclusion in the same cultures. P-values <0·05 were considered statistically significant when compared to controls by Students t-test.

  2. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin.

% Dead cells28·2 ± 4·8620·2 ± 2·1615·1 ± 1·1211·2 ± 1·489·0 ± 1·229·4 ± 0·5217·6 ± 2·8
P 0·0160·0030·0020·0010·0010·009
% Live cells67·4 ± 4·0870·8 ± 3·5681·8 ± 4·1483·8 ± 4·0487·8 ± 2·1789·2 ± 2·7982·4 ± 4·15
P 0·0620·0010·0010·0010·0010·007
Viable cell number146629 ± 123010579600 ± 295136414167 ± 126133277800 ± 99939702750 ± 410121455450 ± 289652725125 ± 155081
P 0·0360·0210·0300·0360·0380·0003

Simultaneous expansion of Mk-P and HPSC made possible by conditions that affect both these progenitor populations

The expansion potential of HSPC and Mk-p from CB MNC was explored in suspension cultures. Progenitor-enriched MNC were isolated on gelatin/ficoll samples from 10 to 20 ml samples of fresh CB and the starting numbers of CD34high HSPC and Mk-p were determined. Following 8–10 d in culture, flow cytometry analysis proved to be a technical challenge due to a large proportion of dead cells derived from the MNC fraction. A systematic analysis was developed using multiple gates and selected markers to identify true Mk -p and HSPC populations and eliminate irrelevant populations (Figure S1). Mk-p were classified as CD45dim-neg/SSClow/CD41high cells (Fig 1A), and HSPC were identified as CD45dim-neg/SSClow/CD34high cells (Fig 2A). The proliferation of the different cell populations was measured as cell numbers that were counted at harvest and the average cell numbers of all experiments were calculated. As the average cell input numbers for the individual progenitor populations were highly variable (Tables II and SII), the fold expansion was calculated in each individual experiment and the average fold expansion of all experiments is presented in Figs 1–2. The early CD41high/CD45dim/neg Mk-p, which were extremely rare on day 0, about 3000/5 × 105 MNC (Table II) underwent a remarkable 3–4-fold (P < 0·02) expansion when grown on FN alone (Table II and Fig 1B) and the CD34high/CD45dim/neg HSPC expanded 6·9–fold (P < 0·01) (Fig 2B and Table II). TPO alone enhanced Mk-p proliferation by 8·3-fold (P < 0·02), in agreement with its known activity (Geddis et al, 2002). TPO alone served as our internal positive control for Mk proliferation. ARP and β-FGF alone or in various combinations, modestly stimulated proliferation of the Mk-p population in the absence of FN (Fig 1B and Table II). SCF alone did not stimulate MNC or Mk proliferation and cell numbers were below control levels with no cytokines (data not shown). A synergistic effect was obtained by adding cytokine combinations to FN and FN with TPO produced a 10-fold increase in the average number of cells and a 25-fold increase in the average fold expansion of Mk-p (Table II and Fig 1B).

image

Figure 1.  FN, TPO and ARP drive expansion of Mk-p and early Mk-p subpopulations detected by high resolution flow cytometry. MNC were cultured for 8–10 d with or without fibronectin and the indicated growth stimulators. At the termination of cultures cells were counted, stained with fluorescent antibodies and analysed by flow cytometry. The initial gate (R1) was set to include only the live non-granular cells (A). A second gate (R2) was therefore set which included only the CD45dim/neg SSClow cells, better known as the blast hole progenitors (Shapiro, 2003). Megakaryocyte progenitors lack CD45 expression (Tomer, 2004), while their precursors can express dim levels of CD45 similar to common myeloid progenitors (Matsumura-Takeda et al, 2007). This gate excluded the non-HSPC and non-Mk cells such as the myelo-monocytic cells (SSChigh) and lymphoid cells (CD45high) that underwent expansion, and mature haematopoietic cells that may have acquired extraneous CD41 labelling in the culture as described in the supplemental Figure S1. The CD45dim/neg SSClow CD41high cells were considered MK-p. The average fold expansion in the number of Mk-p in five or more separate experiments (mean ± SE) is presented in B. All expansion cultures in B demonstrated significant differences in the number of Mk-p compared to day 0 (P ≤ 0·05). The graphs in C and D represent the calculated expansion of the subpopulations of Mk-p that also express CD34 (C) and the myeloid marker CD33 (D) within the Mk-p gate (as demonstrated in the inserts) in at least five experiments. *P < 0·05. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin; SCF, stem cell factor; FGF, β- fibroblast growth factor; CON, control.

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image

Figure 2.  FN and ARP Enhance Expansion of Early Myeloid Progenitors. The flow cytometry gating scheme for detection of CD34+ HSPC in cultures is illustrated in A. The graphs present the average fold expansion measured as live cells that were SSClow CD34high in five or more separate experiments (mean ± SE). The subpopulations of these haematopoietic progenitors expressed myeloid marker CD33 (B) or high levels of CD41, implying megakaryocytic commitment (D). Graphs E and F demonstrate the expansion of non-committed early HSPC having low levels of CD41 (E) with a portion of these cells expressing CD33 (F). FN alone or ARP alone or FN + TPO supported HSPC proliferation. Note that ARP with FN stimulated a >400-fold expansion of the CD34+/CD41low uncommitted HSPC which was partially accounted for by the CD33+ compartment within this population *P < 0·05. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin; SCF, stem cell factor; FGF, β- fibroblast growth factor; CON, control.

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Table II.   Proliferation of the different cell populations.
Growth conditionsNumber of CD45dim/low/SSClow/CD41high MK-p/5 ×105 MNC ± SDExpected average number of MK-p/20 ml CBPotential number of platelets/20 ml CBNumber of CD45+/SSClow/CD34high HSPC/5 ×105 MNC ± SDExpected average number of HSPC/20ml CBCFU-GEMM/20 ml CB (n)CFU-GEMM fold increaseCFU-GM 20 ml CB (n)CFU-GM fold increaseCFU-MK/20ml CB (n)CFU-MK fold increaseTotal myeloid CFU/20ml CB (n)
  1. Cord blood mononuclear cells were cultured (0·5 × 106/ml) in tissue culture flasks coated or uncoated with human fibronectin with 10% autologous plasma and the indicated growth supplements added every 3 d. Cell counts were determined microscopically using a haemocytometer or with a Coulter Counter LH500 automated analyser (Beckman-Coulter Inc) and viability monitored by fluorescent propidium iodide stain or by trypan blue dye exclusion. Progenitor populations were analysed by high resolution flow cytometry as described in Methods and cell numbers were compared to the initial populations at day 0 and significant differences of at least five experiments for each condition was determined by Students t-test.

  2. Following primary liquid cultures the proliferation of megakaryocyte colony-forming units (CFU-MK), myeloid granulocyte macrophage CFU (CFU-GM) and multilineage progenitor cells CFU (GEMM) were assessed in secondary culture colony assays using 2 × 105 cells under appropriate growth conditions for each lineage and counted after 10 d. The table presents colony numbers or the fold increase (±SEM) in the number of colony forming progenitors grown on 10 d culture compared to day 0. Data represent the averages of 3–7 separate experiments harvested on days 8–10. *P < 0·05 (anova test).

  3. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin; SCF, stem cell factor; β-FGF, β- fibroblast growth factor.

  4. *P < 0·05; **P < 0·005; ***P < 0·0005; t-test versus input D0.

  5. < 0·05; ††< 0·005; test versus TPO, D10.

  6. < 0·05; ‡‡< 0·005; test versus TPO + FN, D10.

Input D03082 ± 31711 232 8003·7 × 1096444 ± 49492 456 0003400 ± 3200 6240 ± 440 2800 ± 3200 12480·0
Output D10            
Control1919 ± 3622767 6002·3 × 1095111 ± 16972 044 400445 ± 2260·13 ± 0·07668 ± 3490·11 ± 0·08807 ± 4280·29 ± 0·131920
FN9367 ± 6375*3 746 8001·1 × 101026 340 ± 25 350*10 536 000148 ± 1070·04 ± 0·03384 ± 2090·06 ± 0·05156 ± 840·06 ± 0·03688
TPO15317 ± 7820**6 126 8001·8 × 101011 248 ± 42274 499 2006192 ± 54531·80 ± 1·7012 342 ± 4550**1·97 ± 1·03*5264 ± 889**1·77 ± 0·28*20 684
ARP4214 ± 3523†1 771 6005·3 × 10919 245 ± 6815*7 698 0808256 ± 5762·4 ± 0·19*5884 ± 20860·94 ± 0·473351 ± 370*1·20 ± 0·1210 242
β-FGF4429 ± 64401 771 6005·3 × 10915 790 ± 74836 316 0003514 ± 24521·02 ± 0·772688 ± 11980·43 ± 0·272677 ± 14520·96 ± 0·458879
SCF     8491 ± 71712·468 ± 2·2423 042 ± 6075·233·69 ± 1·38*4983 ± 18981·780 ± 0·5936 516
TPO + SCF13 445 ± 10842**5 378 0001·6 × 10107401 ± 30722 960 4006236 ± 4091*1·813 ± 1·2717 232 ± 4470**2·76 ± 1·103936 ± 19061·406 ± 0·5927 403
TPO + ARP19 185 ± 27493*7 674 0002·3 × 101011 929 ± 4308*4 771 6006567 ± 4320*1·909 ± 1·356755 ± 21951·03 ± 0·491949 ± 12180·696 ± 0·5415 271
TPO + β-FGF11 707 ± 101494 682 8001·4 × 10109918 ± 27393 967 2002852 ± 16970·829 ± 0·5315 105 ± 81482·42 ± 1·8515 804 ± 4992*5·644 ± 1·633 762
FN + ARP859 ± 785343 6001 × 10915 672 ± 12 9806 268 8003440 ± 32000·91 ± 0·333240 ± 44000·38 ± 0·651913 ± 11160·683 ± 0·3411 593
FN + TPO34 077 ± 44 445**13 630 8004·1 × 101014 248 ± 40035 699 20010 428 ± 7920*3·03 ± 2·488694 ± 44011·39 ± 1·007070 ± 3174*2·525 ± 0·9926 192
FN + TPO + ARP389 667 ± 342 053***,†,‡155 866 8004·7 × 101118 906 ± 7089*7 562 28029 240 ± 19 233*,†8·50 ± 6·0163 552 ± 30 538**10·18 ± 6·49*7605 ± 2349*2·716 ± 1·04100 397
FN + TPO + β–FGF255 853 ± 311 232**,‡102 341 2003·1 × 10118014 ± 18293 205 60022 017 ± 18 9176·40 ± 5·1935 412 ± 14 620**5·67 ± 3·23*10 582 ± 3909*3·779 ± 1·2268 012
FN + TPO + SCF286 770 ± 28 351***,†,‡114 708 0003·4 × 10119629 ± 2271*3 851 60023 886 ± 14 836*6·94 ± 4·6322 416 ± 8113**3·59 ± 1·84*7590 ± 2552*2·711 ± 0·8053 892

Acetylcholinesterase readthrough peptide, the stress associated haematopoietic peptide derived from acetylcholinesterase, when added to FN and TPO, was even more powerful, driving a highly significant increase in the number of Mk-p (Table II) with an average 60-fold expansion of this population (P < 0·001) (Fig 1). A poor effect on the number of Mk-p was observed when ARP added to FN alone. ARP was previously shown to robustly stimulate proliferation of CD34+ HSPC (Deutsch et al, 2002; Pick et al, 2006) and megakaryocytopoiesis from CD34+ cells (Grisaru et al, 2006), and to replace SCF as an accessory factor for stimulating proliferation of early haematopoietic progenitor cells (Grisaru et al, 2001). These results defined FN as a potent growth-stimulating agent of Mk-p and demonstrated for the first time that FN alone was capable of stimulating megakaryocytopoiesis, and that this expansion was further enhanced by TPO and ARP, SCF or β-FGF (Table II). To further resolve the progenitor subpopulations that contributed to Mk-p proliferation, the subsets of Mk-p cells, which expressed either CD34 (Fig 1C), or the myeloid marker CD33 (Fig 1D) were analysed. While the entire CD41+ Mk population comprised 1·1% (±0·3) of the fresh CB fraction, the early subsets of CD41high/CD34high Mk-p and the CD41high/CD33 comprised 0·18% (±0·14%) and 0·27% (±0·15%) respectively. FN alone was a powerful stimulator of early Mk-p, doubling the number of CD41high/CD34high cells (P < 0·05) while TPO alone yielded a threefold increase in the number of CD41high/CD34high immature progenitors (Fig 1C and Table II). The combination of TPO with cytokines, in the absence of FN, was not effective in expanding these rare early progenitors, probably due to cytokine-driven differentiation. When progenitor and stem cell adhesion was enabled by coating the plates with FN, TPO supported higher proliferation, which was further enhanced by SCF (Fig 1C). Surprisingly, FN with ARP alone did not drive significant proliferation of CD41high/CD34high cells, probably due to rapid Mk maturation, yet when TPO was added to FN and ARP a fourfold increase was noted.

In contrast, the CD41high/CD33+ Mk-p, a subset with flow cytometric properties similar to clonogenic GEMM progenitors, was found to proliferate autonomously, similar to the common myeloid progenitor (Akashi et al, 2000) and underwent at least two replications with a 5·5-fold increase without cytokine supplementation (Fig 1D). Additionally, FN alone or TPO, ARP or β-FGF reinforced a consistent robust expansion of this early myeloid progenitor compartment. Growth stimulator combinations of TPO and ARP or β-FGF, without FN, facilitated a 30–40-fold expansion, indicating that different adhesive requirements may influence the proliferation of these cells. The combination of FN + TPO with either ARP, SCF or β-FGF stimulated a considerable (30–50-fold) proliferation of CD33+ Mk-p, a population which could provide both myeloid and megakaryocytic cells post-transplant (Fig 1D). Calculation of the platelet-producing potential of these progenitors demonstrated that these cell expansions were clinically relevant. Even if each progenitor did not proliferate further but developed into a single Mk, cells grown with FN + TPO + ARP may produce as many as 4·7 × 1011 platelets from the MNC of 20 ml CB (Table II).

FN and ARP stimulate proliferation of CD34+ HSPC

Considering that expansion of Mk-p requires sufficient numbers of HSPC that can undergo commitment to the Mk lineage, followed by Mk-p expansion we examined the ability of CD34+ progenitor cells and their subsets CD34high/CD33+ or CD34high/CD41+ low and high subpopulations to undergo self-renewal and differentiation under different growth conditions. Similar analytical steps used for assessing Mk-p were used to resolve HSPC expansion. After elimination of dead and nonspecifically labelled cells a CD45dim/SSClow gate was set to include all cells in the progenitor area ‘blast hole’. These cells were further analysed for high CD34 and low or high CD41 or CD33 expression.

The addition of FN alone was the most potent single stimulator of CD34+ HSPC (Table II), with an average of 6·9-fold expansion above baseline (P < 0·05) (Fig 2A). Additionally all cultures that contained ARP maintained a high proliferation of HSPC, even higher than treatment with TPO, confirming previously published data that ARP protects and drives CD34+ HSPC and early myeloid cell proliferation (Deutsch et al, 2002; Grisaru et al, 2006; Pick et al, 2006). To further identify the subpopulations contributing to HSPC expansion in these cultures, the CD34+/CD33+ (Figs 2B, E) or CD34+/CD41high committed Mk-p (Fig 2C and Table SII) or CD34+/CD41low early uncommitted HSPC (Fig 2D and Table SII) subsets were analysed. FN alone stimulated the CD34+/CD41high Mk-p and the addition of ARP to FN did not result in net accumulation of these progenitors, probably due to ARP-driven maturation of committed Mk-p (Guimaraes-Sternberg et al, 2006). The addition of TPO to FN + ARP produced a synergistic proliferative effect, again demonstrating the capacity of ARP to drive robust proliferation of uncommitted HSPC and TPO to stimulate proliferation of Mk-p. The CD34+/CD41low cells, located within the CD45 blast hole (data not shown), which have been described as uncommitted HSPC, rather than bona fide Mk-p, with GEMM-like qualities (Akashi et al, 2000; Shapiro, 2003), proliferate without the addition of cytokines. These rare early precursors (found at a frequency of 1/104 MNC, Table SII) are vigorously stimulated by the addition of any single cytokine and even more so by cytokine combinations. FN alone yielded an 81-fold increase in the number of CD34+/CD41low cells, while ARP alone increased this population by ninefold. The addition of ARP to FN stimulated a dramatic 442-fold increase of these uncommitted cells. The GEMM-like CD34+ CD41low/CD33+ subpopulation was stimulated 45·6-fold by FN and the addition of ARP to FN increased the proliferation of these progenitors by 65-fold, although the addition of TPO to FN was less effective, probably due to mature Mk pull out. The addition of β-FGF to TPO and FN supported both committed CD34+/CD33+ and uncommitted CD34+/CD33+/CD41low myeloid progenitors (Figs 2B, E and Table SII). Adding ARP to TPO and FN produced fewer CD34+ CD41low/CD33+ cells than any of the conditions, supporting the idea that ARP drives differentiation of CD41low uncommitted progenitors to become CD41high committed progenitors and to further mature into Mk. These data support the notion that FN plays an essential role in HSPC expansion, and that both TPO and ARP are involved in proliferation and maturation of Mk.

FN enhances cytokine stimulated Mk and HSPC colony forming progenitors

The effect of FN on the clonogenic capacity of the HSPC was assessed as CFU-Mk, CFU-GM and multilineage progenitor cells (GEMM) after 10 d of primary cultures grown with FN and the indicated growth stimulators (Table II). We calculated similar numbers of CFU-Mk and CFU-GM as those previously reported (Kanamaru et al, 2000) which were both expanded with TPO or SCF alone. No significant differences in the multipotential GEMM colonies were obtained due to the great response variability between individual CB units. ARP was the only single cytokine able to elicit a robust proliferation of GEMM, confirming previously reported data (Grisaru et al, 2001). The effect of FN on lineage-committed and multilineage progenitors was evident when FN was combined with other growth modulators. There was an approximate threefold increase in the number of CFU-Mk and a 5–8 fold increase in the number of CFU-GEMM when combined with TPO or TPO + SCF, β-FGF, or ARP (Table II).

FN increased number of total Mk and stimulates their maturation

The capacity of FN to increase the total number of Mk and stimulate their maturation was assessed. The expansion of Mk was measured as live CD41high/CD14 cells including the higher SSC mature MK. Including cells with higher SSC required elimination monocytes that bind CD41+ platelets via FC receptors in culture (Harding et al, 2007). Anti-CD14 antibodies were used to eliminate nonspecifically labelled cells (Figure S1).

Fibronectin alone produced a fivefold (P < 0·05) Mk expansion, and TPO alone a threefold expansion (P < 0·015). The combination of TPO and FN enhanced expansion better than either factor alone (P < 0·05). FN and ARP did not facilitate proliferation of Mk, probably due to accelerated maturation and demise of committed Mk-p in the presence of ARP, confirming the results obtained with CD41high/CD34high Mk-p or CD34high/CD41high early Mk-p. This hypothesis was also supported by increased maturation and high apoptosis levels (Fig 4). TPO + FN + ARP and TPO + FN + SCF were found to stimulate more than a sixfold expansion of mature Mk (P < 0·03), reconfirming the notion that ARP can replace SCF in culture (Grisaru et al, 2001) (Fig 3). As the relative proliferation of progenitors was higher than that of total Mk, it appears that the mature Mk were quickly driven to terminal differentiation and platelet production (Fig 4D).

image

Figure 4.  FN with TPO or ARP enhance megakaryocyte maturation. Polyploid cells produce higher distinct fluorescent peaks which are proportional to the amount of DNA. The MK population was gated as CD41high cells (A). Ploidy compartments of these cells are clearly visible as dot plots (B) or as histogram peaks (C). TPO stimulated the MK cell towards the 4N phase, ARP stimulated the Mk cell towards the 16N stage and up to the fully mature 64N stage (D). The graph (E) presents expression of ITGA2B mRNA in the cultures. Graphs illustrate the in the average fold increase of the day 10 ratio of ITGA2B/GAPDH divided by the ratio of ITGA2B/GAPDH of day 0. The highest increase in ITGA2B expression (sevenfold) was noted when TPO and ARP were added to the FN containing cultures. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin; SCF, stem cell factor; FGF, β- fibroblast growth factor; CON, control.

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image

Figure 3.  Expansion of megakaryocytes. Fold expansion of the total Mk population was assessed by flow cytometry as total live CD41high/CD14 without the progenitor gate. The graphs present the average fold expansion measured as live cells in five or more separate experiments (mean ± SE). It is noteworthy that the combination of TPO + FN + ARP was found to be the most potent stimulator of Mk expansion, *P < 0·05. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin; SCF, stem cell factor; FGF, β- fibroblast growth factor; CON, control.

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To confirm the maturation of Mk in culture, the CD41high Mk were gated and ploidy analysed by staining with PI, which enabled relative quantification of the DNA. (Figs 4A–C) and the percentage of cells in each ploidy compartment (Fig 4D). TPO stimulated Mk DNA to 4N (which are either polyploid and or proliferating 2N-G2 Mk, while ARP drove the Mks to 16N–64N ploidy (full maturation).

We also investigated the expression of the platelet-specific fibrinogen receptor ITGA2B, known to be upregulated during endomitosis, with its level being a function of Mk maturation (Raslova et al, 2007). The combination of FN with TPO and ARP yielded a maximal expression with 6·5-fold increase (Fig 4E; P < 0·05), once again supporting the notion that FN and ARP drive robust Mk maturation. A threefold increase in expression was reached with FN + TPO (P < 0·04). In the absence of FN, TPO + ARP stimulated a 2·8-fold increase (P < 0·05) and ARP alone stimulated a 3·2-fold increase (Fig 4E).

Discussion

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

While CB provides an alternative source of HSPC for allogeneic transplantation in children, several obstacles have prevented its full potential as a reliable source of haemopoietic stem cells in adults. Two main limitations are: (i) the availability of sufficient numbers of haemopoietic stem cells to support engraftment of all lineages in the adult (Ballen et al, 2007; Brunstein et al, 2007a,b) and (ii) the low absolute number of Mk progenitor cells and their poor engraftment, even in young children, which results in prolonged severe thrombocytopenia (Kanamaru et al, 2000). Multiple platelet transfusions remain the only current treatment (Brunstein et al, 2007a), thus new strategies to increase cell dose are needed. Therapeutic modalities that combine ex vivo expanded progenitor cells and unmanipulated CB are being explored and recently been shown to be safe (Kelly et al, 2009). The time to platelet engraftment was shown to correlate with the amount of transplanted early Mk CD34+ CD41+ cells after allo-BMT (Begemann et al, 2002) providing a reasonable basis for expanding transplantable Mk-p, although the optimal expansion conditions are still not known. Expanding CD34+ HSPC from CB is not practical due to the indispensable low numbers of stem cells in the graft (Brunstein et al, 2007b). Therefore, we explored the feasibility of developing an efficient method for expansion of progenitors from a small aliquot of whole CB, in short term cultures of progenitor-enriched MNC using new and improved conditions that partially mimicked the BM haematopoietic niche.

To assess true expansion of HSPC and Mk-p in culture it was essential to develop a high resolution FACS protocol for the identification of these rare cells and their subpopulations. A systematic hierarchal analysis was developed using appropriately selected sequential gates to enable fine tuned phenotype analysis of HSPC and Mk-p while eliminating other CD41+ myeloid cells. After eliminating dead cells from the analysis, which was critical in cultures initiated with MNC, a second gate was set to include only the SSClowCD45dim/neg cells. In contrast to mature myelo monocytic and lymphoid cells, which express robust CD45 levels, the Mk lineage lacks CD45 expression (Tomer, 2004; Forsberg et al, 2006), while its precursors can be CD45dim, similar to other haematopoietic progenitor cells (Matsumura-Takeda et al, 2007). Thus Mk-p were characterized as live SSClow/CD45dim/neg/CD41high cells. This approach excluded from analysis the non-Mk cells (myeloid, lymphoid and monocytes) that underwent expansion or that acquired external CD41 labelling from adherent platelets or microparticles during culture (Harding et al, 2007).

Most fresh CB MNC which die during the first week in culture secrete molecules that damage the proliferating progenitors. Therefore the first step to enable expansion of Mk-p was to create an optimal survival environment using FN-coated flasks and new growth factors known to drive megakaryocytopoiesis. FN, the adhesive platform in the hematopoietic niche, is known to support viability and proliferation of HSPC, via integrin-α4β1, α5β1-dependent adhesion and signalling and to affect Mk maturation and proplatelet formation in the BM (Schick et al, 1996; Yokota et al, 1998)(Avecilla et al, 2004; Balduino et al, 2005). Here we demonstrated that FN alone enhanced the survival of CB MNC and afforded a viability advantage to HSPC and Mk-p, simultaneously in agreement with the known positive effect of FN on the viability and engrafting potential of these cells in the BM (Sagar et al, 2006). Furthermore, the Mk-p and CD34+ HSPC, which are notably scant on day 0, underwent a dramatic expansion when grown on FN. FN also escalated proliferation of the CD34high/CD41lhigh/Mk-p and the CD34high/CD41low and CD34high/CD41low/CD33 myeloid progenitor cells. The CD34+/CD41low cells were CD45dim, located within the blast hole (data not shown). These cells are similar in phenotype to GEMM like cells (Akashi et al, 2000), proliferate autonomously, yet are increased 100-fold with FN, introducing FN as a potent growth stimulating agent for both the Mk-p and early common myeloid progenitor subpopulations, which can be further expanded under appropriate conditions. FN alone had no effect on CFU-Mk, indicating that the clonogenic progenitors require more than adherence to FN or a single signal to proliferate.

Healthy haematopoietic BM niches provide both FN adhesive support and sufficient levels of TPO for normal megakaryocytopoiesis and platelet production. TPO was shown to support survival of very early HSC expressing low levels of the receptor, to trigger proliferation of progenitors that express high levels of Mpl and drive their more responsive progeny to terminal maturation (Paulus et al, 2004). Many studies have shown that TPO induces proliferation of CD34+, CD41+ and CD34+/CD41+ cells (Piacibello et al, 2000; Paulus et al, 2004; Kaushansky, 2005; Deutsch & Tomer, 2006), The addition of Flt-ligand, IL-6 and IL-11 to TPO were shown to increase leucocyte expansion with differentiation and terminal maturation into the Mk lineage with high numbers of immature CD34+ CD41+ Mk progenitor cells reported (De Bruyn et al, 2005). Furthermore, engagement of the integrin-α4β1, as is the case with FN enhances TPO-induced megakaryopoiesis (Fox & Kaushansky, 2005). Our results support these findings. We have now demonstrated that, similar to the healthy haematopoietic niche, FN and TPO provided an excellent platform for human Mk-p proliferation ex-vivo, enhanced the growth of all Mk-p and maturation of Mk. SCF in our studies as previously reported by others, was not a stimulator of CB-derived Mk and therefore not included in many experiments and the addition of SCF to TPO did not provide any advantage over TPO confirming previous studies by others (De Bruyn et al, 2005).

The addition of the haematopoietic ARP peptide, derived from acetylcholinesterase further stimulated proliferation of both early HSPC and Mk-p and maturation of late Mk-p. This combination was more potent in expanding the CD34+ HSPC and the early CD41low progenitor populations than the addition of either SCF or β-FGF. Both TPO and ARP are stress/inflammation responsive plasma proteins known to stimulate Mk-p engraftment and Mk maturation and human platelet production in non-obese diabetic/SCID mice (Grisaru et al, 2006; Pick et al, 2006). The combination of these factors may preferentially accelerate Mk-p maturation and platelet production in the BM following stress. Indeed, transgenic mice overexpressing ARP have elevated progenitor numbers and platelet counts (Grisaru et al, 2001) and ARP can drive rapid maturation of megakaryoblasts (Guimaraes-Sternberg et al, 2006). Here we report normal human Mk maturation under the influence of ARP. Fewer Mk-p and their subpopulations were observed when ARP was added to FN without TPO, indicating that following adherence of Mk-p to FN, accelerated maturation occurred (Fig 5). Surprisingly, fewer Mk-p of the myeloid phenotype were observed with TPO or ARP in the presence of FN than in the absence of FN, indicating that accelerated maturation of the CD33+ subpopulation may be facilitated by FN with either TPO or ARP (Grisaru et al, 2001, 2006). Facilitation of myeloid cell maturation by FN has been recently reported (Esendagli et al, 2009). Additionally, different adhesive properties may regulate the proliferation of these cells in the BM. With FN and TPO, SCF and β-FGF had almost identical effects on proliferation of the CD45dim/neg/SSClow/CD41high population, indicating that the addition of either ARP or SCF to FN and TPO drove proliferation and a maturation pullout of the CD34+ cells. This notion was confirmed by the robust proliferation of CD33+ Mk-p population and stimulation of CFU-Mk obtained when either ARP or SCF were added to FN and TPO, validating our previous report that SCF can be replaced by ARP as an accessory growth factor (Grisaru et al, 2001). Additionally, all ARP-containing cultures maintained higher proliferation of HSPC, and myeloid CFUs than cultures with TPO, in agreement with the known activity of ARP on CD34+ HSPC in vitro (Deutsch et al, 2002; Grisaru et al, 2006; Pick et al, 2006). Yet the addition of ARP to FN did not increase the numbers of Mk-p, once again implicating ARP as a potent Mk maturation factor as previously reported (Guimaraes-Sternberg et al, 2006). ARP drives vigorous proliferation of uncommitted HSPC and the addition of ARP to FN + TPO further stimulated their growth as well as all of their progeny, particularly the CD34+/CD41lowuncommitted highly proliferative myeloid progenitors. These results, similar to others (Begemann et al, 2002), support the notion that transplantation of adequate numbers of CD34+ CD41+ cells could enable continued extensive proliferation in the patients due to synergy with high level TPO present in the thrombocytopenic patients. Fig 5 summarizes the synergistic activities of these growth stimulators. FN, TPO and ARP also drive simultaneous Mk maturation, seen as increased numbers of high ploidy cells and increased expression of GPIIb/IIIa. These data demonstrated the potent effect of FN, TPO and ARP on early myeloid progenitor proliferation, and Mk maturation conditions that occur simultaneously in the haematopoietic niche under stress. We are currently investigating whether Mks triggered by FN with TPO and ARP lead to increased proplatelet formation and platelets release.

image

Figure 5.  Megakaryocytopoiesis is best facilitated by a FN platform with ARP for CD34+ multilineage HSPC expansion and TPO to drive megakaryocyte progenitor expansion and MK maturation. Fibronectin, a major component of the bone marrow haematopoietic niche, protects CD34high CD41low progenitors from cell death thus enabling further maturation or more robust proliferation. ARP drives proliferation of CD34high cells and maturation of committed CD41high megakaryocyte progenitors. Proliferation of megakaryocyte progenitors is further stimulated by thrombopoietin. The combination of these factors stimulates each level of development required for optimal expansion and maturation of megakaryocytes from stem cells. ARP, acetylcholinesterase readthrough peptide; TPO, thrombopoietin; FN, fibronectin.

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In summary, using FN as a growth platform and new factors which included ARP, we have defined the conditions for expanding well resolved subpopulations of platelet and myeloid producing progenitors. FN protects HSPC and committed Mk-p by preventing their demise, thus enabling further expansion and maturation under optimal conditions. While the addition of TPO supports the proliferation of all Mk-p populations, ARP affects the uncommitted and subpopulations. The addition of ARP to FN and TPO creates an optimal microenvironment for HSPC and Mk-p expansion ex-vivo. Mk-p expanded under these conditions provided an average 60-fold increase. We calculate that from only 20 ml of CB one should be able to obtain 18 × 107 Mk-p (1·6 × 105 Mk-p/ml × 20 ml × 60). If calculated by average cell numbers (Table II and Table SII), we obtained a very similar number, 15 × 107 CD45dim-neg/SSClow/CD41high Mk-p. Even if the Mk progenitors only mature and do not proliferate, an individual Mk has the potential to produce about 3000 platelets, subsequently increasing the potential platelet production to 2·70 × 1011 platelets. These Mk-p could supply and additional 50 × 109 platelets/litre blood in an transplanted adult with an average blood volume of 5 l. We assume that these ex vivo expanded MK–p will mature and have the potential to supply enough platelets to facilitate earlier recovery from severe thrombocytopenia and reduce the risk of bleeding after CBT. Further studies are warranted to examine the benefit of these conditions for facilitating more rapid neutrophil and platelet recovery in severely myelosuppressed patients following CBT.

Acknowledgements and support

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

We gratefully acknowledge the expert technical assistance of Mrs Shoshana Baron. This work was supported by the Biodisc programme of the German Federal Ministry of Education and Science (BMBF) (V.R.D.) and a research grant from the Israel Ministry of Health (Chief Scientist) (V.R.D.).

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  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements and support
  7. References
  8. Supporting Information

Fig S1. Enrichment of CD34+HSPC and CD41+ Mk-p.

Table SI. Enrichment of hematopoietic cell populations following isolation of fresh cord blood MNC on gelatin/ficoll–paque.

Table SII. Early progenitor cell expansion in liquid cultures.

Appendix S1. Enrichment of HPSC and MK-p from MNC from CB samples.

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