PRODUCTION OF MEGAKARYOCYTES IN BIOREACTORS

Abstract

An in vitro process for producing megakaryocytes, and optionally platelets from the megakaryocytes, including the steps of cultivating stem cells, e.g. induced pluripotent stem cells, to generate aggregated pluripotent stem cells, preferably cultivating the aggregates in suspension in medium, inducing differentiation in these aggregates, and isolating megakaryocytes from the culture medium.

Claims

1. Process for producing megakaryocytes from stem cells (SC) which are at least pluripotent, the process comprising the steps of a) cultivating the stem cells to generate cultivated stem cells, b) cultivating the cultivated stem cells, c) cultivating the aggregates comprising the cultivated stem cells in suspension in medium with agitation to produce cultivated aggregates comprising stem cells, d) differentiating the cultivated aggregates comprising stem cells to megakaryocytes by adding differentiation factors to the cultivated aggregates comprising stem cells, and e) separating megakaryocytes from the cultivated aggregates in order to obtain the megakaryocytes.

2. Process according to claim 1, wherein the stem cells are induced pluripotent stem cells (iPSC).

3. Process according to claim 1, wherein step c) and step d) are carried out concurrently.

4. Process according to claim 1, wherein in step b) the stem cells are contacted in suspension with micro-carriers to generate aggregates comprising or consisting of stem cells and micro-carriers, and that in step c) the aggregates comprising or consisting of stem cells and micro-carriers are cultivated in suspension in medium with agitation to produce cultivated aggregates comprising stem cells and micro-carriers.

5. Process according to claim 1, wherein in step e) the megakaryocytes are separated from the stem cell-aggregates, respectively from the stem cell-micro-carrier-aggregates.

6. Process according to claim 1, wherein in step b) the stem cells are incubated under cell culture conditions for a static phase for at least 12 h, followed by cultivation with agitation.

7. Process according to claim 1, wherein the micro-carriers are non-porous.

8. Process according claim 1, wherein the micro-carriers are coated with a protein promoting adherent growth.

9. Process according to claim 1, wherein step g), comprises irradiating megakaryocytes or platelets with at least 5 Gy.

10. Process according to claim 1, comprising lysing the megakaryocytes and/or platelets to generate a lysate from the megakaryocytes and/or platelets, wherein the lysate is for use in a medical treatment.

11. Process according to claim 1, wherein the process is free from other cells and is free from human serum and free from animal serum.

12. Serum-free megakaryocytes obtained by a process according to claim 1.

13. Serum-free platelets obtained by a process according to claim 1.

14. Serum-free megakaryocytes according to claim 12 for use as a medicament in the treatment of thrombocytopenia, platelet refractoriness, or for use in the treatment of alloimmunization to HLA or alloimmunization to human platelet antigens, or for use in the treatment of a pregnant woman, or for use in the treatment of a transplant patient.

15. Serum-free platelets according to claim 13 for use as a medicament in the treatment of thrombocytopenia, platelet refractoriness, or for use in the treatment of alloimmunization to HLA or alloimmunization to human platelet antigens, or for use in the treatment of a pregnant woman, or for use in the treatment of a transplant patient.

16. Use of serum-free platelets according to claim 13 as a substitute for fetal calf serum.

17. Serum-free megakaryocytes according to claim 14 lysed to generate a lysate, for use in the treatment of alloimmunization to HLA or alloimmunization to human platelet antigens, or for use in the treatment of a pregnant woman, or for use in the treatment of a transplant patient.

18. Process according to claim 1, wherein step b) comprises contacting the cultivated stem cells with micro-carriers, to generate aggregates comprising or consisting of stem cells and micro-carriers.

19. Process according to claim 1, further comprising: f) cultivating the megakaryocytes under conditions for generating platelets from megakaryocytes, and further optionally isolating the platelets, and g) irradiating the megakaryocytes and/or platelets.

20. Process according to claim 8, wherein the protein promoting adherent growth is fibronectin and/or laminin.

Description

[0042] The invention is now described in greater detail by way of examples and with reference to the figures, which show in

[0043] FIG. 1 micrographs of aggregates of iPSC/megakaryocyte-progenitors and micro-carriers, at A at the end of step c) and at B at the end of step d) of the process,

[0044] FIG. 2 A, B graphic displays of the yield of megakaryocytes from the process,

[0045] FIG. 3 at A results of flow cytometry, at B and E fluorescence micrographs, at C, D, F, G light micrographs of megakaryocyte aggregates produced in the process, without micro-carriers (upper row, B, C, D) and of aggregates of iPSC/megakaryocyte-progenitors with micro-carriers produced in the process (lower row, E, F, G),

[0046] FIG. 4 A, B a graphic display of analytical results for megakaryocyte markers, and in

[0047] FIG. 5 A, B micrographs of platelets produced in vitro from megakaryocytes produced according to the invention, without irradiating the megakaryocytes (A, left) or following irradiating of the megakaryocytes (B, right).

EXAMPLE 1: PRODUCTION OF MEGAKARYOCYTES USING MICRO-CARRIERS

[0048] As an example for PSC, the human iPSC line (hCBiPSC2) was cultivated in conditions without feeder cells and without serum components, giving feeder-free and xeno-free conditions. These cells were seeded at 50000 cells/cm.sup.2 on 12 non-treated culture plates (Falcon by Corning, USA) coated with human recombinant laminin, LN521 (BioLamina). The iPSC were fed daily with StemMACS (Miltenyi Biotech) medium and passaged 2 to 3 times per week when confluency was reached.

[0049] In this exemplary process, the differentiation of stem cells to megakaryocytes of step b) and the preferred step c) including contacting of the megakaryocytes with micro-carriers are done concurrently. From the culture plates, iPS cells were harvested and suspended at a concentration of 6.25 cells/mL APEL and StemMACS (V/V) or only APEL medium, containing 50 ng/mL BMP4 and 50 ng/mL VEGF, both of which are added both on day 0 and day 2 of this culture. On day 4 and day 8, SCF was added to 50 ng/mL and TPO was added to 50 ng/mL and IL-3 was added to 25 ng/mL for inducing differentiation to megakaryocytes in APEL medium.

[0050] As micro-carriers, animal-product free non-porous polystyrene micro-carriers (SoloHill, PALL, Dreieich, Germany) were sterilized by autoclaving and then coated with laminin by suspending the micro-carriers in 2 μg/mL human recombinant laminin (LN521, BioLamina) in PBS buffer for at least 24 h at 4° C.

[0051] On day 0, PSC were gently mixed at a density of 12.5×10.sup.6 cells per 50 mL medium with added 281.25 mg of the laminin-coated micro-carriers per 50 mL medium and incubated without agitation for approximately 24 h. During this static incubation phase, aggregates of the micro-carriers with the PSC were formed. Then, the suspension was incubated with agitation by stirring at 50 rpm in spinner flasks for at least 12 d prior to isolating megakaryocytes, with exchange of the medium for fresh medium every 2 to 3 days.

[0052] FIG. 1 shows micrographs, at A of the aggregates of the micro-carriers with the PSC on day 0, at B at day 7 of the aggregates of micro-carriers with differentiated iPSC/megakaryocyte progenitors. These pictures show that the cultivation results in a drastic increase in size of the aggregates, indicating growth of the megakaryocytes.

[0053] Megakaryocytes were isolated from the culture supernatant twice a week, starting from day 12 after the addition of the differentiation factors on day 0.

[0054] Analysis of cell size showed that these megakaryocytes had an increased cell size and polyploidy resembling that of human native bone marrow-derived megakaryocytes.

EXAMPLE 2: PRODUCTION OF MEGAKARYOCYTES USING CELL AGGREGATES WITHOUT MICRO-CARRIERS

[0055] In this example, iPSC and megakaryocytes obtained by differentiation from the PSC were cultivated as pure cell aggregates, without presence of suspended micro-carriers. hCBiPSC2 cells that were cultivated in laminin-coated culture plates as described in Example 1 were harvested and seeded into StemMACS medium. Then, the PSC were transferred to animal serum-free synthetic medium APEL (StemCell, Vancouver, Canada) supplemented with 5% protein-free Hybridoma medium II (PFHMII, Gibco by Life Technologies, Darmstadt, Germany). The PSC were incubated in spinner flasks at 50 rpm until aggregates consisting of PSC only were formed. In case, differentiation of the aggregates was observed, the ROCK inhibitor Y-27632 (Calbiochem Novabiochem, Sandhausen, Germany) was added to a concentration of 10 ng/mL medium.

[0056] On day 0 and day 2 of differentiation of the cultivated cell-only aggregates to megakaryocytes, BMP4 and VEGF were added to a concentration each of 50 ng/mL, then on day 4 and day 8 SCF and TPO were added to a concentration each of 50 ng/mL medium and IL-3 was added to a concentration of 25 ng/mL medium. From day 12 onwards, two times per week medium was exchanged for fresh medium containing the SCT and TPO, each at 50 μg/mL. Cultivation was in spinner flasks by stirring at 50 rpm.

[0057] Megakaryocytes were separated from the culture supernatant by the steps of 5 min centrifugation 800 rpm or sedimentation for 30 min.

[0058] FIG. 2 at A shows the total number of megakaryocytes and at B shows the number of megakaryocytes (MK) obtained per initial iPSC cell introduced into the process, obtained according to Example 2 (Cell-aggregates, left-hand column) and obtained according to Example 1 (Cell-MC-aggregates, right-hand column). ** denotes standard deviation p<0.01. FIG. 2 clearly shows that the process using cultivation of aggregates of micro-carriers and megakaryocytes results in a drastic increase in production of megakaryocytes, both in absolute numbers and in relation to initial numbers of PSC that were processed by the process.

[0059] FIG. 3 shows analytical results for the megakaryocytes produced according to Example 1 (Cell-MC-aggregates) and according to Example 2 (Cell-aggregates).

[0060] FIG. 3 A depicts the flow cytometric results of DNA content measurement in megakaryocytes stained with propidium iodide, showing that the megakaryocytes produced according to Example 1 had a higher DNA content compared to the megakaryocytes obtained according to Example 2. This indicates that the process using aggregates of megakaryocytes with micro-carriers generates megakaryocytes of a higher quality than the process using aggregates consisting of megakaryocytes only.

EXAMPLE 3: IRRADIATING MEGAKARYOCYTES

[0061] Megakaryocytes that were produced according to Example 1, using cultivation of aggregates of micro-carriers and megakaryocytes, were isolated from the culture medium and irradiated with 30 Gy

[0062] The irradiated megakaryocytes and an aliquot of the same megakaryocytes that was separated before irradiating were used to generate platelets by cultivating the megakaryocytes under static conditions in APEL medium supplemented with 100 ng/mL TPO and 50 ng/mL SDF-la in the upper chamber of a double chamber bioreactor containing a 2 μm pore membrane for separating the lower chamber. Medium of the same composition was pumped through the lower chamber at a rate of 140 mL/min, and platelets were isolated from the medium exiting the lower chamber.

[0063] The platelets were deposited on a collagen-coated glass and analysed by microscopy after anti-CD61 staining. FIG. 5, left micrograph, shows platelets produced by non-irradiated megakaryocytes, and FIG. 5, right micrograph shows platelets produced by irradiated megakaryocytes. This results shows that functional platelets can be produced by the in vitro process from the megakaryocytes, and that the megakaryocytes can be irradiated without significantly impairing the functionality of the platelets generated by them.

EXAMPLE 4: USE OF MEGAKARYOCYTES AS A MEDICAMENT FOR REPLACING PLATELETS IN VIVO

[0064] As a representative of a human recipient patient, NOD/SCID/IL-2Rγc−/− mice were injected into the tail vein with 3×10.sup.6 megakaryocytes that were produced according to Example 1 or Example 2.

[0065] After 1 h following injection of the megakaryocytes, platelets originating from the injected megakaryocytes were found.