EP4689076A1

EP4689076A1 - Heat-inactivated mesenchymal stem cells and uses thereof

Info

Publication number: EP4689076A1
Application number: EP24712514.9A
Authority: EP
Inventors: Hermanus Johannes Marco Eijken; Anne Louise Schacht REVENFELD; Bjarne Kuno MØLLER
Original assignee: Aarhus Universitet
Current assignee: Aarhus Universitet
Priority date: 2023-03-29
Filing date: 2024-03-22
Publication date: 2026-02-11
Also published as: WO2024200276A1

Abstract

The present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), a process of providing a pre-conditioned media, and the pre-conditioned media obtainable by thereby, uses of HI-MSCs and the pre-conditioned media and the expanded HSC population of cells, for use as a medicament.

Description

HEAT-INACTIVATED MESENCHYMAL STEM CELLS AND USES THEREOF

Technical field of the invention

The present invention relates to a process for expanding stem cells. In particular the present invention relates to heat-inactivated mesenchymal stem cells (HI- MSC) for use as feeder cells in a process of expanding hematopoietic stem cells (HSCs).

Background of the invention

Hematopoietic stem cells (HSCs) are a type of stem cell that can differentiate into all the different types of blood cells in the body. They are responsible for maintaining the constant supply of new blood cells that the body needs to function properly. HSCs are continuously dividing in the body in order to produce new blood cells, which makes them a good candidate for gene editing. However, HSCs can also enter a state of dormancy in which they are not actively dividing. In order to ensure that gene editing is successful, it is important to expand HSCs in the laboratory in a way that promotes their proliferation, or active division. Sternness refers to the ability of a stem cell to maintain its undifferentiated state and to give rise to different types of cells. It is an important property of stem cells because it allows them to continuously regenerate and repair tissues throughout an individual's lifetime.

During HSC expansion, it is possible for HSCs to lose their sternness, or their ability to maintain their undifferentiated state and to give rise to different types of cells. This can occur for a variety of reasons, including the culture conditions used to expand the HSCs, the presence of certain signaling pathways or growth factors, or the accumulation of genetic mutations.

Loss of sternness can be a problem during HSC expansion because it can lead to the differentiation of HSCs into more specialized cell types, which reduces their ability to regenerate and repair tissues. It can also limit the potential clinical applications of the expanded HSCs, such as in bone marrow transplantation or gene editing.

To prevent loss of sternness during HSC expansion, it is important to carefully control the culture conditions and signaling pathways that the HSCs are exposed to, and to regularly monitor the cells for genetic mutations or other changes that could affect their sternness.

One potential use of expanded HSCs is in bone marrow transplantation, a procedure that is used to treat a variety of blood disorders and immune system diseases. In a bone marrow transplant, HSCs are infused into a patient to replace their own damaged or diseased bone marrow. Expanding HSCs in the laboratory allows doctors to obtain a larger number of HSCs for transplantation, which can be especially useful in cases where the patient's own bone marrow is not able to produce enough healthy HSCs.

Another potential use of expanded HSCs is in gene editing. Gene editing is a technique that allows scientists to make precise changes to an individual's genetic material in order to correct genetic defects or to introduce new traits. One of the most commonly used gene editing techniques is called CRISPR-Cas9, which uses a special enzyme called Cas9 to cut the DNA at a specific location, allowing researchers to delete, insert, or replace a specific sequence of DNA. The gene edited cells can then be used to study the effects of gene editing in cell culture or animal models, or potentially to treat genetic diseases in humans.

In order for gene editing to be successful, the cells being edited must be actively dividing. This is because the changes made by the CRISPR-Cas9 system are incorporated into the genome of the cell during DNA replication, which occurs during cell division. If the cells are not dividing, the gene editing will not be successful.

Overall, expanding HSCs for clinical applications and for gene editing has the potential to improve our understanding of blood disorders and immune system diseases, and to develop new therapies for these conditions.

Developments in the past decades improved our knowledge regarding expansion of HSC including the use of mesenchymal stromal/stem cells (MSCs). Co-culturing HSCs with mesenchymal stromal/stem cells (MSCs) has been shown to stimulate in vitro HSC expansion (1, 2). In such culture systems HSCs are seeded on top of a layer of pre-cultured MSCs. It is believed that MSCs stimulate HSC proliferation by direct and indirect interaction including secretion of cytokines and growth factors. Although MSC feeder cells have been successfully used, the development of a standardized culture protocol that include more than one cell type (co- culture) is a complex and challenging task. Issues that complicate co-cultures and thereby clinical implementation include:

A) Growth medium. Growth medium is often optimized for one specific cell type to keep it in the right phenotype and to support proper cell growth. Finding a growth medium meeting the requirement of all involved cell types involves substantial optimization and is often impossible resulting in comprised conditions for one of the cell types.

B) Proliferation of the supporting cells. MSCs are adherent cells and must be pre-cultured to allow for cell attachment in the culture dish prior to the start of the co-culture. During the co-culture, MSCs will continue to proliferate which makes it very challenging to standardize the amount of MSCs in the co-culture. Moreover, as MSCs keep on proliferating during the entire culture period they can reach an amount that will be too high to support a proper cell culture.

C) Contamination of other cell types. Additional purification steps will be required to exclude the presence of the supporting cell type.

D) Timing/Logistics. Logistics of a co-culture are more complex. Prior to the start of the co-culture, MSC need to be isolated and pre-cultured. The start of HSC expansion is therefore depending on the confluency of the MSCs.

E) Additional regulatory requirements. The supporting cells need to be isolated and in vitro expanded. All regulatory requirements need to be fulfilled for the supporting cells on top of the cells of interest.

To eliminate unwanted proliferation of MSCs in HSC-MSC co-cultures, MSC feeder layers have been used to support HSC expansion (3, 4). A feeder layer basically consists of adherent cells that are unable to divide but still maintain the ability to secrete factors that stimulate proliferation. By irradiation or mitomycin C treatment, the proliferation capacity of MSC can be limited while secretion of essential factors is kept intact. Albeit the use of feeder cells eliminates the disadvantages of cell proliferation in co-cultures, the setup is still very challenging regarding the other points addressed above.

HI-MSCs prepared for other applications are normally used directly after heat inactivation. The inventors have previously found that HI-MSC can be stored, for at least two days, in a refrigerator and used directly therefrom (stored HI-MSC). Such cells are specifically identified in PCT Application No. PCT/EP2022/076148, published as WO 2023/046709.

Hence, an improved expansion process of HSCs would be advantageous, and in particular a process that provides an expanded HSC population in the correct growth medium with less contamination, at a lower cost, and in a shorter time would be advantageous.

Summary of the invention

Thus, an object of the present invention relates to the optimization of HSC expansion protocols. An improved expansion process of HSCs would be advantageous, and in particular a process that provides an expanded HSC population in the correct growth medium with less contamination, at a lower cost, and in a shorter time would be advantageous.

In particular, it is an object of the present invention to provide an expanded HSC population of cells that solves the above mentioned problems of the prior art with

A. growth medium,

B. proliferation of the supporting cells,

C. contamination of other cell types, and/or

D. timing/logistics.

The present invention has surprisingly solved these problems by the use of heat- inactivated MSCs, which in contrast to the known inactivated cells, are metabolically inactive, and were found to be able to support expansion of HSCs in diverse setups, such as from freely-diffusible co-cultures to pre-conditioned media with no transfer of intact HI-MSC cells.

Further particular advantages of using HI-MSC are at least:

• that less amounts of MSCs are needed, since a surprisingly low amount of HI-MSC can induce expansion of the HSC cell pool, no prior expansion of the MSC cells are needed, and there is no risk of later proliferation of the MSC cells,

• that cells can be stored for longer (at various temperatures) since the heat-inactivated cells are no longer metabolically active and does not require immediate use after heat inactivation, thus the cells can be prepared in advance as a "ready-to-use" solution,

• that there is no risk of transferring minute amounts of mitomycin C to patients receiving the expanded HSC cultures and/or other factors needed for regular maintenance of non-HI-MSC cells,

• that administration of HSCs to the patient is a monoculture, without any risk of administrating HI-MSCs to the patient, and/or

• that the expanded cell population has an improved viability, whilst maintaining their differentiation capabilities, i.e. sternness.

These advantages are thus provided by the following aspects:

In a first aspect, the present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI- MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs.

In another aspect, the present disclosure relates to a process of providing a pre-conditioned media as described in the first aspect, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media. In another aspect, the present disclosure relates to a process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

In another aspect, the present disclosure relates to a pre-conditioned media obtained by or obtainable by the process as described above. In a related aspect, the present disclosure relates to a container comprising the pre-conditioned media. In another related aspect, the present disclosure relates to a use of the pre-conditioned media, to expand a population of HSCs.

In another aspect, the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as cells for pre-conditioning HSC growth media.

In another aspect, the present disclosure relates to a kit comprising the preconditioned media as described herein, and optionally comprising instructions for expanding HSCs.

In another aspect, the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as feeder cells for expanding HSCs.

In another aspect, the present disclosure relates to a HSC population of cells obtained by or obtainable by the process as described herein. In a related aspect the present disclosure relates to a container comprising the HSC population of cells as described herein.

In another aspect, the present disclosure relates to a kit comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.

In another aspect, the present disclosure relates to the HSC population of cells as described herein, for use as a medicament. In a still other aspect, the present disclosure relates to the HSC population of cells as described herein, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease. In another such aspect the present disclosure relates to a method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease in a patient in need thereof, the method comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.

Brief description of the figures

Figure 1 shows staining pattern of 7AAD changes after heat-inactivation. The level of 7AAD staining was determined immediately before (pre-HI) and after (post HI, DO) heat-inactivation and after storage at 4 C° for 2 and 3/4 days. N=2.

Figure 2 shows the concentration of stored HI-MSC remains stable. The concentration of HI-MSC was determined using quantitative flow cytometry. The intact cells were identified based on forward scatter (FSC) and side scatter (SSC) properties, excluding only cellular debris. N = 3.

Figure 3 shows HI-MSC induce dose-dependent increase of proliferation of CD34+ HSC in culture. Four different concentrations of HI-MSC used as an expansion support to CD34+ HSC. The total amount of viable CD34+ cells after 6 days of culture in HSC monoculture was compared to the number of viable CD34+ HSC in co-cultures with HI-MSC at different concentrations. Mean ± SD. N=4.

Figure 4 shows HI-MSC support high viability of CD34+ HSC in culture. Assessment of cellular viability by 7AAD staining followed by flow cytometric analyses at baseline (DO) and after 6 days of culture in either monoculture or coculture with different HI-MSC concentrations. Mean ± SD. N=4.

Figure 5 shows HI-MSC support dose-dependent generation of CD34+ HSC with a relevant stem cell phenotype. The total number of CD34+ cells with associated stem cell marker (CD90, CD38, CD133, CD201) was determined by flow cytometric analyses at baseline (DO) and after 6 days of culture. Mean ± SD.

N=4. Figure 6 shows more pronounced HSC over HI-MSC donor effect on the expansion of CD34+ HSC in co-cultures. CD34+ HSC from four different donors were each co-cultured with four HI-MSC batches for 6 days. The total amount of viable CD34+ cells in the co-cultures was divided by the number from the corresponding HSC monoculture. N=4.

Figure 7 A and B shows HSC donor variation has larger impact on amount of relevant CD34+ HSC than HI-MSC donor variation. After 6 days, the stem cell phenotype of cultured CD34+ HSC was determined by flow cytometry. The total number of CD34+ HSC expressing a certain marker is grouped for each HSC donor, showing the level obtained in HSC monoculture and that from the cocultures with four different HI-MSC batches. N=4.

Figure 8 shows HI-MSC prevents drop in cellular viability of cultured CD34+ HSC. The cellular viability was evaluated by 7AAD staining at baseline (DO), on D3 and D6 of culture. The 7AAD- are shown for each of the time points for culturing of CD34+ HSC from four different stem cell donors, using the same four HI-MSC batches for each HSC donor. N = 4.

Figure 9 shows HI-MSC provides similar expansion support to CD34+ HSC with and without UM171. After 6 days of culture, the total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. N = l.

Figure 10 shows HI-MSC boosts viability of cultured CD34+ HSC. The cellular viability was evaluated with 7AAD staining of HSC at baseline (DO) and after 6 days of culture. The percentage of 7AAD- cells are reported as the viability. N = l.

Figure 11 shows HI-MSC increases production of CD34+ HSC with relevant stem cell phenotype. The absolute number of viable CD34+ HSC with an associated stem marker is provided for baseline and after 6 days of culture in the HSC monoculture and in the co-cultures. N = l. Figure 12 shows HI-MSC facilitated expansion support of CD34+ HSC is contact - independent and can also be obtained with HI-MSC primed HSC medium. Different CD34+ HSC monocultures were prepared and compared to co-cultures with HI- MSC. HI-MSC primed HSC medium was incubated for 24h prior to culture setup, using the same HI-MSC concentration as the co-cultures. Monocultures with preincubated HSC medium served as control to HI-MSC primed medium. For the contact-independent co-culture, CD34+ HSC and HI-MSC were physically separated by a membrane with 0,4 pm pores, allowing the passage of small molecules and vesicles. After 6 days of culture, the total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. Mean±SD. N=3.

Figure 13 shows HI-MSC and their derivatives support optimal CD34+ HSC viability. The cellular viability was evaluated with 7AAD staining of HSC at baseline (DO) and after 6 days of culture. The percentage of 7AAD- cells are reported as the viability. Mean±SD. N = 3.

Figure 14 shows HI-MSC and HI-MSC derivatives induce similar stem cell phenotype in cultured CD34+ HSC. The absolute number of viable CD34+ HSC with an associated stem marker is provided for baseline and after 6 days of culture in the HSC monocultures and in the co-cultures. To enable comparison across all culture setups, the amount of cells detected in the contact-independent cultures were volume adjusted to match the lower volume in the other cultures. Mean±SD. N = 3

Figure 15 shows priming of HSC medium with different concentrations of HI-MSC yields similar effect, when used as HSC expansion support in identical final concentration. HSC was primed with two concentrations of HI-MSC (60.000/mL and 2xlO'^K6/mL) for 24h. Both were used as expansion support for CD34+ HSC in a final concentration corresponding to 20.000 HI-MSC/mL.

A) After 6 days of culture, the total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. B) The stem cell phenotype of cultured CD34+ HSC was determined by flow cytometric analysis at D6 of culture. The total number of CD34+ HSC with an associated marker is reported. N = l.

Figure 16 shows expansion of CD34+ HSC alters both number and type of colonies in CFU assay. After 6 days of culture, the differentiation potential of expanded CD34+ HSC was evaluated in an in vitro CFU assay. After 14 days of differentiation, the cells were evaluated by flow cytometric analysis.

A) The number of colonies identified obtained with and without expansion.

B) The colony distribution, based on type of colony. The colonies were identified based on expression of CD14, CD15, and CD235a. N = l.

Figure 17 shows HI-MSC remain stable in culture, both as a monoculture and coculture with CD34+ HSC.

A) HI-MSC was diluted to a final concentration of 20.000 HI-MSC/mL in HSC medium and incubated for 6 days after which the concentration was determined by quantitative flow cytometry. Three independent experiments.

B) Co-culture of 20.000 HI-MSC/mL and 50.000 CD34+HSC/ml was performed. After 6 days of culture, the concentration of HI-MSC was determined by flow cytometric analysis. Mean±SD. N=4.

Figure 18 shows that a clinically relevant HSC medium supports CD34+ expansion in both mono- and co-culture setting. The RUO medium, StemSpan SFEMII, was replaced by the GMP compliant StemSpan AOF.

A) After 6 days of culture, the total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. N = l.

B) The total number of CD34+ cells with associated stem cell marker (CD90, CD38, CD133, CD201) was determined by flow cytometric analyses at baseline and after 6 days of culture. N = l.

Figure 19 shows that the type of inactivation method affects plastic adherence and morphology of MSC. Immediately after inactivation (OH), either with heat (HI- MSC) or with irradiation (irr. MSC), cells were seeded in culture flasks containing MSC medium. MSCs from the same batch, which was inactivated, were seeded in parallel and served to demonstrate the expected adherence and morphology behavior of non-inactivated MSC. Adherence and morphology was additionally evaluated 24H and 72H post-inactivation. N = l.

Figure 20 shows different viability stain patterns and apoptosis levels observed for MSC inactivated with heat and irradiation. A) The level of 7AAD staining was evaluated before inactivation of MSCs and at different time points after. Mean ± SD. N = l. B) The fractions of live cells (Annexin- PI-), apoptotic cells (Annexin + PI-), and necrotic/dead cells (Annexin+ PI+) were determined before inactivation of MSCs and at different time points after. N = l.

Figure 21 shows that metabolic activity is maintained after irradiation of MSC, but not after heat inactivation. The metabolic activity was evaluated before inactivation of MSC and at different time points after inactivation with either irradiation or heat. Mean ± SD. N = l.

Figure 22 shows that freshly prepared and pre-frozen (-80 °C) HI-MSC primed medium provides similar CD34+ HSC expansion support. Monocultures of CD34+ HSC with or without addition of primed medium from HI-MSC were prepared. One version on the HI-MSC-primed medium was freshly prepared, while another had been stored for -80 °C for 9 days prior to usage. A co-culture of CD34+ HSC and HI-MSC was also created. All cultures had duration of 6 days. A) The total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. B) The cellular viability was evaluated with 7AAD staining of cultured HSC. The percentage of 7AAD- cells is reported as the viability. N = l.

Figure 23 shows the stem cell phenotype of cultured CD34+ HSC. After 6 days of culture, the absolute number of viable CD34+ HSC with an associated stem marker was determined. N = l.

Figure 24 shows that concentration and 7AAD staining of long-term stored HI-MSC remain stable. A) The concentration of HI-MSC was determined using quantitative flow cytometry. The intact cells were identified based on forward scatter (FSC) and side scatter (SSC) properties, excluding only cellular debris. B) The level of 7AAD staining was determined at immediately after inactivation and following storage at 4 C° for 17 and 21 months. N=l.

Figure 25 shows HI-MSC stored for 21 months preserve supportive capabilities on HSC expansion. A) After 6 days of culture, the total number of viable CD34+ HSC was determined by flow cytometry and related to the seeded number of CD34+ HSC at DO, yielding the expansion fold. B) Assessment of cellular viability by 7AAD staining, followed by flow cytometric analyses after 6 days of culture. C) The total number of CD34+ cells with associated stem cell marker (CD90, CD38, CD133, CD201) was determined by flow cytometric analyses after 6 days of culture. N = l.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Mesenchymal Stem Cells" or NSC

The terms "Mesenchymal Stem Cell" or"MSC", as used herein, refers to adult progenitor cells that can self-renew and can differentiate into multiple lineages such as osteoblasts, adipocytes, and chondroblasts. MSCs can be isolated from numerous tissues such as bone marrow, adipose tissue, the umbilical cord, liver, muscle, and lung. MSCs adhere to plastic when maintained under standard culture conditions. MSCs express CD73, CD90, and CD105, but under standard culture conditions lack expression of CD31, CD45, CDllb, and CD19 surface molecules.

Heat-inactivated cells

In the present context, the terms "Heat-inactivated cells", "Heat-inactivated- Mesenchymal Stem Cell", "HI-MSC cells" or "HI-MSC" is to be understood as a heat-treated metabolically inactive cell that has maintained its cellular integrity. A heat-inactivated cell is not viable anymore.

The heat treatment can for example be conducted as provided in example 1.

In the present context, the terms "cellular integrity" or "maintained their cellular integrity" is to be understood as i) the metabolically inactivated cells have a clear cellular structure when viewed under a light microscope (data not shown), ii) the metabolically inactivated cells stain positive for a nuclear staining such as 7-Aminoactinomycin D (7-AAD).

In the present context, the terms "viability", or "cell viability", is a measure of the proportion of live, healthy cells within a population of cells. Viability can be measured in different ways. In the present disclosure 7AAD staining is applied, which is a standard measure of viability, in which viable cells are not stained, and thus 7AAD negative (7AAD-), due to an intact cell membrane.

In the present context, the term "metabolically inactivated" refer to cells which are without mitotic and metabolic activity.

In an embodiment, the metabolically inactivated MSCs are metabolically inactivated and cannot reduce MTT to formazan. In another embodiment the metabolically inactivated MSCs are shown to be metabolically inactive, such as through the use of an MTT- or XTT assay, such as CyQUANT XTT Cell Viability assay (Thermo Fischer Scientific).

In a further embodiment, the metabolically inactivated MSCs cannot adhere to plastic. In a further embodiment, the metabolically inactivation of the MSCs is irreversible.

In the present context, "Hematopoietic stem cell", "HSC", "Hematopoietic stem cells" or"HSCs" is understood as the rare population of multipotent cells residing in the bone marrow and other hematopoietic tissues that have a unique ability to self-renew and differentiate into all blood cell lineages, including red blood cells, leukocytes, and platelets, throughout the lifespan of an organism. HSCs are essential for maintaining the homeostasis of the hematopoietic system and for replenishing the blood cell pool after injury or infection.

HSCs express a variety of differentiation markers that distinguish them from other hematopoietic cell types. CD45, CD34, CD90, and CD133 are commonly used to identify and isolate HSCs, while HSCs with low or no CD38 expression may have enhanced self-renewal capacity and the ability to differentiate into multiple lineages. These cells are also thought to be more quiescent and resistant to chemotherapy.

CD201, also known as endothelial protein C receptor (EPCR), is a transmembrane glycoprotein that is expressed on the surface of various cell types, including endothelial cells, hematopoietic cells, and some stem cells. As provided by the examples, CD201 may be induced by the inclusion of UM171 in the HSC growth medium. The expression of CD201 is currently investigated in HSCs.

Pre-conditioned medium

In the present context, the terms "Pre-conditioned medium", or "primed medium" are used interchangeably herein.

Balanced salt solution (BSS)

In the present context, balanced salt solution (BSS) is a solution made to a physiological pH and isotonic salt concentration. Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. Examples of balanced salt solutions which may find use with the present invention are:

• Isotonic saline;

• Alsever's solution;

• Earle's balanced salt solution (EBSS);

• Gey's balanced salt solution (GBSS);

• Hanks' balanced salt solution (HBSS);

• (Dulbecco's) Phosphate buffered saline (PBS);

• Puck's balanced salt solution;

• Ringer's balanced salt solution (RBSS); • Simm's balanced salt solution (SBSS);

• TRIS-buffered saline (TBS); and

• Tyrode's balanced salt solution (TBSS).

Preferably: Ringer, Isotonic saline, Albumin supplemented sodium chloride composition. Additives may include antibiotics, vasoconstrictors, growth factor, salt, sugars, or other stimulants.

The present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), a process of providing a pre-conditioned media, and the pre-conditioned media obtainable by thereby, uses of HI-MSCs and the pre-conditioned media and the expanded HSC population of cells, for use as a medicament. These aspects, and more, will now be described in additional detail.

Expansion of hematopoietic stem cells (HSCs),

As provided by the examples, the inventors have surprisingly realized that the use of HI-MSCs, were found to be able to support expansion of HSCs in diverse setups. The HSCs can either be expanded by

• supplementing their growth medium with a "pre-conditioned" medium, wherein a HSC growth medium has been contacted with HI-MSCs and the HI-MSCs have been removed afterwards,

• by adding HI-MSCs directly into a container comprising HSC growth medium and the HSCs to be expanded, or

• by adding HI-MSCs into a container having separate chambers, a first chamber comprising HSC growth medium and the HSCs to be expanded and a second chamber comprising HSC growth medium and the HI-MSCs, the chambers being separated from each other by a membrane having pores allowing for a non-cellular diffusion between the two chambers,

In a first aspect, the present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the preconditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs.

The process should continue as long as needed for the HSCs to be induced to expand, thus the HI-MSC cells or the pre-conditioned growth medium may be removed, however, it is vital that expansion of the HSCs has been induced. Thus, in one embodiment of the present disclosure, the time sufficient to expand the HSCs is at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.

As introduced above, the present invention can be performed in several different variations. A variant is to conduct a "classical" co-culture system where the cells are only in fluid connection, and thus cannot directly contact each other. In one embodiment of the present disclosure, the HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell co-culture, such as a non-contacting co-culture.

The fluid connect is often established by the use of specific filters, that allows for a free dissociation of particles under a specific size, as shown by the examples the present inventors have conducted their experiments using 0,4 pm separation, however the skilled person will be able to select other filters, with the same expected result. In one embodiment of the present disclosure, the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.

Another variation to the setup can be to place the cells in direct contact. Thus, in another embodiment of the present disclosure, the HI-MSC cells are placed in spatial contact with the HSCs.

The examples in the present application are conducted with 12-well plates, however, especially when the process is introduced into a GMP compliant environment, where the output is for use in a clinical setting, the working volumes will be significantly larger. The skilled person can use different measures to arrive at the number of cells to be used, and will in general scale- up the process in accordance to the volume or the surface used. Since the cells are in suspension, a volumetric measure may be preferred, and thus in a preferred embodiment, 20.000 HI-MSC/mL are provided. In another embodiment, between 10.000-500.000 HI-MSC/mL are provided such as providing 30.000 HI-MSC/mL, such as 50.000 HI-MSC/mL, such as 80.000 HI- MSC/mL, such as 100.000 HI-MSC/mL, such as 200.000, such as even providing 400.000 HI-MSC/mL. Ideally, a ratio is selected corresponding to a ratio of 0.4: 1 - 10: 1 (HI-MSC:HSC). In one embodiment of the present disclosure, at least 1000 HI-MSC cells are provided, such as at least 5000, such as at least 10,000, such as at least 20,000 HI-MSC cells are provided, such as even up to 6*10⁶ HI-MSCs, such as even 12*10⁶ HI-MSC cells are provided. In one embodiment of the present disclosure, between 40.000/cm² and 70.000/cm² HI-MSC cells are provided, such as 50.000/cm², such as even 60.000/cm² HI-MSC cells are provided, preferably 60.000/cm² HI-MSC cells are provided. In one embodiment of the present disclosure, at least 1000 HSCs are provided, such as at least 5000, such as at least 10,000, such as at least 20,000, such as at least 30,000, such as at least 40,000, such as at least 50,000 HSCs are provided.

A good measure on the amount of HI-MSCs that should be provided, is the relative amount of HI-MSCs in relation to HSCs. As presented by the examples, in particular example 2, the process functions over a wide range of ratios, since as low as 4000 HI-MSC in relation to 50000 HSCs provide a sufficient expansion, and even up to 100000 HI-MSC in relation to 50000 HSCs also provide expansion. In one embodiment of the present disclosure, at least 5% HI-MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b.

HI-MSC may be prepared by various methods known to the skilled person. Freshly harvested MSC were centrifuged at 440 x g for 5 min. and resuspended in MEM-a to reach a concentration of 3-5x10^6 cells/mL. The cell suspension was added to sterile 1,5 mL tubes (max 1 mL in each) and heat-inactivated at 50 °C for 35 min. Heat inactivation was stopped by cooling the tubes in ice bath for 5 min. Cell concentration and viability were evaluated, using flow cytometry. Each HI-MSC batch was stored at 4 °C and used at day 3-4 post-inactivation, in StemSpan SFEM II (STEMCELL Technologies) or saline with 2-5% human serum albumin (HSA) at a final concentration of approximately 5x10^6 HI-MSC/ mL. Prior to use in co-cultures, the concentration and degree of 7AAD staining was determined for each HI-MSC batch, using flow cytometry.

MSCs can be inactivated by different means of heat treatment. The cells may be inactivated by heating to a temperature in the range 40-75°C, such as 40-60°C, preferably at 45-55°C, more preferably at about 50°C, for a period from 5 minutes to 2 hours, 10 minutes to 1 hour, preferably such as 15-45 minutes, more preferably for a period of about 30 minutes or such as at least 10 minutes at 45-75°C. In some embodiments, the heat inactivation may be stopped by cooling the tubes in ice bath, such as for around 2-10 minutes, preferably around 5 minutes.

MSCs can be provided from different tissues. Thus, in an embodiment, the MSCs are selected from the group consisting of adipose derived MSCs, human umbilical cord MSCs, bone marrow derived MSCs, dental pulp MSC and induced pluripotent mesenchymal stem cells, preferably the MSCs are adipose derived MSCs.

In a preferred embodiment, the MSCs are adipose derived or bone marrow derived. The invention is applicable to both these types of MSCs, showing that the method is generally applicable to MSCs.

In another embodiment, the adipose derived MSCs are derived from a stromal vascular fraction (SVF).

The MSCs may be modified in different ways before being inactivated. Thus, in an embodiment, the MSCs are primed and/or pretreated MSCs and/or genetically modified MSCs. Examples of priming or pre-treatment are i) incubation with cytokines, interleukins, growth factors such as VEGF or other secreted factors such as damage-associated molecular patterns (DAMPs), ii) pharmacological or chemical agents, iii) exposure to hypoxic conditions, iv) exposure to other cells types such as injured endothelial cells v) expansion of MSCs in 3D conditions.

Thus, in an embodiment, the priming/pretreatment is selected from the group consisting of incubation with factors such as TNFalpha, INFgamma, ILlbeta, and damage-associated molecular patterns (DAMPs), incubation with pharmacological or chemical agents, exposure to hypoxic conditions, exposure to other cells types such as injured endothelial cells, and expansion of MSCs in 3D conditions.

In yet an embodiment, the MSCs are derived from a mammal, and preferably a human being.

In a further embodiment, the MSCs are expanded (such as 1-8 passages) or nonexpanded MSCs.

In an embodiment, the MSCs are thawed cryopreserved mesenchymal stem cells or freshly harvested mesenchymal stem cells. Both options work equally well.

Inactivation can be performed in different types of liquids (media). Thus, in an embodiment, the MSCs are inactivated in a liquid, such as saline or a balanced salt solution (BSS), such as PBS. BSS are considered pharmaceutical acceptable mediums. In yet an embodiment, the media comprises carrier protein such as human serum albumin (HSA), such as 0.5%-20% HSA, such as 0.5%-10% HSA, such as 0.5% to 5% HSA, such as 1-3% HSA by weight.

HSCs can be derived from multiple different sources, and the process as described herein will be able to expand the HSCs, no matter the source of origin. In many clinical aspects, the source providing the highest amount of HSCs for expansion is where HSCs are chemically mobilized from bone marrow and released into the peripheral blood. When the HSCs are present in the peripheral blood, the cells are subsequently obtained therefrom. Such a mobilization can for instance be done by the use of Plerixafor. In one embodiment of the present disclosure, the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood. Likewise, the HI-MSCs can be derived from multiple different sources. In one embodiment of the present disclosure, the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived. In a specific embodiment of the present disclosure, the adipose derived MSCs are derived from a stromal vascular fraction (SVF).

As presented in the background, expansion of HSCs are needed for multiple different purposes, and thus in some instances, other up-stream and/or downstream processes (such as expansion/gene-editing/enrichment) are performed prior to and/or after contact with the HI-MSCs. In other embodiments, the HSCs are manipulated further after they have been expanded. In one embodiment of the present disclosure, the HSCs have been genetically modified prior to, during, and/or after expansion, such as by the use of CRISPR-Cas9.

In one embodiment of the present disclosure the expanded HSCs are to be used in bone marrow transplantation. In one embodiment of the present disclosure, the expanded HSCs are to be used in gene editing, such as CRISPR-Cas9. The gene edited cells can then be used to study the effects of gene editing in cell culture or animal models, or to treat genetic diseases in humans.

In one embodiment of the present disclosure, the MSCs are derived from a mammal, and preferably a human being. The mammals may also be commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds.

The MSCs themselves may also have been manipulated in different ways, before or after their inactivation. In one embodiment of the present disclosure, the MSCs are expanded MSCs.

The examples, in particular example 2 and example 7, furthermore show the surprising stability of the HI-MSCs, and thus show the cells can be stored at different temperatures. In one embodiment of the present disclosure, the HI-MSC cells are provided directly after heat inactivation, or the HI-MSC cells have been stored at e.g. below 10°C such as below 5°C, such as in the range 10°C-0.1°C, between -24°C and 5°C, or have been stored at -80°C, such as having been cryogenically stored. In one embodiment of the present disclosure, the HI-MSC cells have been stored at 4 °C and used 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation.

A particular advantage of using HI-MSCs is that the risk of injecting contaminating compounds is reduced. In one embodiment of the present disclosure, the HI-MSC and/or the HSC medium is free from chemical cell inactivation agents, such as mitomycin C.

Another advantage of using HI-MSCs is that a precise feeder: responder cell ratio can be employed. Since HI-MSC remain intact in culture, this ratio can be easily adjusted over time as well, when HSC expand. This provides the ability to have a more standardized cell-to-cell ratio than with other types of feeder cells.

The HI-MSCs are not able to actively secret compounds to their surrounding medium, and the HI-MSCS are for instance metabolically inactive. In one embodiment of the present disclosure, the HI-MSC cells are metabolically inactivated.

The HI-MSC can maintain their cellular integrity for sustained periods of time. In one embodiment of the present disclosure, the provided HI-MSCs in step a) has maintained their cellular integrity, such as maintained their cellular integrity

- for at least 2 days such as at least 15 days, such as at least 30 days or such as at least 60 days, such as at least 4 months, when stored in a temperature range between 0.1 and 10°C, such as 1-8°C, such as 2-6°C, such as 3-5°C, or such as around 4°C; and/or for at least 2 days, such as at least 7 days, such as at least 15 days, such as at least 30 days, such as at least 60 days, when stored in a temperature range 11-28°C, such as 15-28°C, or such as 18-25°C. As seen by the examples, in particular example 11, an upper limit to the storage has currently not been identified. As such, cells can be stored for at least 17 or even 21 months, and maintain their cellular integrity as well as their function as feeder cells when stored in a temperature range between 0.1 and 10°C, such as 1-8°C, such as 2-6°C, such as 3-5°C, or such as around 4°C. As such, cells can even be stored for years, such as 1 years post inactivation, such as even 2 years post inactivation.

In some embodiments, the cells are not stored for more than 3 years, such as not more than 4 years, such as not more than 5 years.

To properly expand HSCs, and provide expanded HSCs it is vital that a HSC growth media is used, so that the expanded HSCs are as similar as possible to endogenous HSCs. Thus, preferably one or more of the components rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, are used in the HSC growth media, to expand the HSCs. As shown by the examples, in particular example 5, UM171 can be left out of the HSC growth medium when HI-MSC are applied to expand HSCs. HI-MSC thus supports similar expansion fold and viability of cultured CD34+ HSC both in the presence and absence of UM171 in the HSC growth medium. The expression of CD201 was completely absent, when UM171 was removed from the HSC medium.

A slightly different stem cell phenotype arises, when UM171 is not applied in coculture, yet substantial amounts of CD34+ HSC with a relevant phenotype is still produced. Thus, in one embodiment, all the components rhSCF, rhTPO, rhFLT3L, rhIL-6, and UM171 are added to the HSC growth medium, whilst in another embodiment UM171 is not used, thus only rhSCF, rhTPO, rhFLT3L, and rhIL-6 are added to the HSC growth medium.

Antibiotics are most often also applied in the process, and preferred options may be streptomycin and/or penicillin, however the skilled person will know that the process is not influenced by the choice of antibiotics.

As also presented by the examples, such as example 8 in relation to the other examples, the process functions independently of the choice of HSC growth media applied, in other embodiments, the HSC growth media may be StemSpan SFEM II or StemSpan AOF. In one embodiment of the present disclosure, the HSC growth media comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.

Process of providing a pre-conditioned media

As presented in the above, the HSCs can be expanded by supplementing their growth medium with a "pre-conditioned" medium. Thus, the process of providing such a pre-conditioned media is also provided, as well as its different uses. The process may either provide the pre-conditioned media to be used in the first aspect of the disclosure, or the process may provide the preconditioned medium for any further use. In another aspect, the present disclosure relates to a process of providing a pre-conditioned media as described in the first aspect, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

In another aspect, the present disclosure relates to a process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

In one embodiment of the present disclosure, the media is selected from

- a growth media, such as a HSC growth media; and a liquid cell medium, such as a balanced salt solution (BSS), such as PBS, preferably isotonic saline and/or a pharmaceutical acceptable composition.

The HSC growth media is described in more detail above, such as StemSpan SFEM II or StemSpan AOF, as well as the different components the skilled person may add. In one embodiment of the present disclosure, the HSC growth media is supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF. However, especially when the preconditioned media is prepared in advance, such as for bulk storage or when offered for sale it may be advantageous to leave out the additional components, and only add one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, when the pre-conditioned add the additional components. In even further embodiments, the skilled person may find it advantageous to leave out these components entirely, since the pre-conditioned media might be added in minute amounts, i.e. when the process of the first aspect is ongoing the HSC growth medium provided in step b, will comprise the correct amounts of rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin and/or penicillin.

The pre-conditioned medium can be prepared using various amounts of HI-MSC. In one embodiment of the present disclosure, at least 60.000 HI-MSC/mL medium is added, such as up to 2x10^6 HI-MSC/mL medium, preferably 2x10^6 HI- MSC/mL medium.

In one embodiment of the present disclosure, further comprising supplying a container comprising HSCs with an amount of the pre-conditioned media, sufficient to expand the HSCs.

In another aspect, the present disclosure relates to a pre-conditioned media obtained by or obtainable by the process as described above. In a related aspect, the present disclosure relates to a container comprising the pre-conditioned media as described above. As shown in example 10, a pre-conditioned medium prepared as described above can be stored at -80 °C prior to usage. As such, batches may easily be prepared and stored for long periods of time. The skilled person would not expect any further degradation to occur by prolonged periods of -80 °C storage.

Uses, population of cells, kits and medical uses

The processes for expanding HSCs and obtaining a HI-MSC pre-conditioned medium, have now been introduced. The present disclosure relates to several further aspects, including but not limited to different uses of HI-MSCs, expanded HSCs, and pre-conditioned media.

In another aspect, the present disclosure relates to a use of the pre-conditioned media as described above, to expand a population of HSCs.

In another aspect, the present disclosure relates to a HSC population of cells obtained by or obtainable by the process as described herein.

It is essential that the HSC cells provided herein are positive for the CD45, and CD34 markers. Further additional markers will also often be present. As presented by the examples, in particular example 5, the inclusion of UM171 in the HSC growth medium results in a cell that is positive for the CD201 marker, and resultantly the exclusion resulting in a lack of CD201 expression. Thus, in one embodiment of the present disclosure, the cells are positive for the markers CD45, CD34 and

• positive for at least one of the markers selected from the group CD90, CD133, and CD201; and/or

• negative for CD38.

In another embodiment of the present disclosure, the cells are positive for the markers CD45, CD34, and positive for at least one of the markers selected from the group CD90, and CD133, and/or negative for CD38 and CD201.

As seen by example 4, the viability of CD34+ HSC is markedly higher in HI-MSC co-cultures compared to HSC monocultures after 6 days of culturing. By measuring the viability on DO, D3, and D6 of the culture, it was possible to identify that HI-MSC seemingly reduces a drop in CD34+ HSC viability, as measured in D3 (Figure 8). Thus, in one embodiment of the present disclosure, the HSC population of cells has an increased viability, when compared to a HSC monoculture.

Especially for transport- and storage purposes, the HSC population of cells may be comprised in a contained, thus in another aspect, the present disclosure relates to a container comprising the HSC population of cells as described herein.

Kits may also be provided comprising the components to conduct the processes as described herein, thus in another aspect, the present disclosure relates to a kit comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.

Likeworthy, the pre-conditioned media may also be provided in a kit, thus in another aspect, the present disclosure relates to a kit comprising the preconditioned media as described above, and optionally comprising instructions for expanding HSCs.

As the overall goal of the present disclosure is to provide clinically relevant cells, the present disclosure also provides the uses of these cells as medicaments and/or treatments of various diseases. Thus, in another aspect, the present disclosure relates to the HSC population of cells as described herein, for use as a medicament. A related aspect is thus, the HSC population of cells as described herein, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukaemia, or an immune system disease.

A likewise related aspect, is the method of treatment of such disease. Thus the present disclosure also relates to a method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukaemia, or an immune system disease in a patient in need thereof, the method comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.

The cells find use in treatments of all types of leukemias, blood disorders, and immune system diseases, however, some limited groups can be mentioned. In one embodiment of the present disclosure, the leukemia is a leukemia selected from the group consisting of Multiple myeloma (MM), Myelodysplastic syndrome (MDS), Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), Hodgkin lymphoma, and Non-Hodgkin lymphoma (NHL), including: Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma, Burkitt lymphoma, and T-cell lymphoma. In one embodiment of the present disclosure, the blood disorder is a blood disorder selected from the group consisting of sickle cell disease, thalassemia, aplastic anemia, and Fanconi anemia. In one embodiment of the present disclosure, the immune system disease is an immune system disease selected from the group consisting of severe combined immunodeficiencies (SCID), chronic granulomatous disease (CGD), Wiskott-Aldrich syndrome (WAS), hemophagocytic lymphohistiocytosis (HLH), severe autoimmune diseases and selected inherited metabolic disorders.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples. Examples

Example 1: Generation of HI-MSC and storage

Aim

To generate heat-inactivated mesenchymal stromal cells (HI-MSC), as stable metabolically inactive entities that can serve as expansion support to HSC.

Materials and methods

Isolation of mesenchymal stromal cells from human adipose tissue

In total 4-6 mL lipoaspirate was washed by PBS and processed by enzymatic digestion in combination with mechanically disruption using collagenase (Nordmark Biochemicals-N0002779) and the GentleMACS Octo Dissociator with C- tubes (Miltenyi). After digestion the solution was centrifuged and the pellet (stromal vascular fraction) was transferred into a T175 culture flask containing 20 mL culture medium and incubated at 37 °C and 5% CO2. The cell culture medium consisted of Minimum Essential Medium (aMEM) (Gibco-Thermo Fisher Scientific), 5% PLTGold Human Platelet Lysate (Mill Creek Life Sciences), 2 mM L-Glutamin (Gibco-Thermo Fisher Scientific) and 50 U/mL Penicillin-Streptomycin (Gibco- Thermo Fisher Scientific). When the T175 culture flask reached 90% confluency, the culture was split using 6 mL TrypLE™ Select (Gibco-Thermo Fisher Scientific) and seeded in new T175 culture flasks. AD-MSCs at first, second, or third passage were harvested using TrypLE™ Select (Gibco-Thermo Fischer Scientific) at 90% confluency and cryopreserved using CryoStor CS10 (Stemcell Technologies) at - 80°C.

Cultivation of MSC

After storage at -80 °C, cells were thawed in a 37 °C water bath and transferred to pre-warmed MSC medium, consisting of MEM- a, supplemented with L- Glutamine, 50 U/mL Penicillin-Streptomycin, and 5% PLTGold human platelet lysate. After centrifugation at 440xg for 5 min, cells were resuspended in MSC medium and seeded in T175 flasks, using approximately 1x10^6 cells/flask. The cells were cultured in a CO2-incubator at 37 °C and 5% CO2 until 80-90% confluence was reached. Subsequently, cells were harvested, initially with a double wash with PBS, followed by detachment using TrypLE. Pre-warmed MSC medium was added, and cell concentration and viability (7AAD staining) were evaluated, using flow cytometry. Heat-inactivation of HI-MSC

Freshly harvested MSC were centrifuged at 440 x g for 5 min. and resuspended in MEM-a to reach a concentration of 3-5x10^6 cells/mL. The cell suspension was added to sterile 1,5 mL tubes (max 1 mL in each) and heat-inactivated at 50 °C for 35 min. Heat inactivation was stopped by cooling the tubes in ice bath for 5 min. Cell concentration and viability were evaluated, using flow cytometry. Each HI-MSC batch was stored at 4 °C and used at day 3-4 post-inactivation, in StemSpan SFEM II (STEMCELL Technologies) or saline with 25 human serum albumin (HSA) at a final concentration of approximately 5x10^6 HI-MSC/ mL. Prior to use in co-cultures, the concentration and degree of 7AAD staining was determined for each HI-MSC batch, using flow cytometry.

Cell counts and viability measurements

Live MSC or HI-MSC were stained with pre-diluted 7AAD (1:5 in PBS) for 5 min. The acquisition of stained cells was performed on a NovoCyte 3000 (Agilent Technologies), using the NovoExpress Software (version 1.5.0, Agilent Technologies). See Materials and methods section in Example 2 for more details. Technical triplicates were measured and the mean value for the absolute number and the fraction of 7AAD+ and 7AAD- cells were used.

Results

The level of 7AAD staining is normally used as a measure of cell viability and as such viable cells are not stained, and thus 7AAD negative (7AAD-), due to an intact cell membrane. Here, we show that the level of 7AAD- cells rapidly drops immediately after heat-inactivation and is further reduced 3-4 days post-heat inactivation (Figure 1). This indicates that the HI-MSC are dead and have a permeable cell membrane.

Moreover, the concentration of the stored HI-MSC at 4 C° remained stable, as evaluated by the total number of intact cells present in the stored cell suspension (Figure 2).

Conclusion

It is possible to generate HI-MSC with a high 7AAD+ fraction that remains stable during storage at 4 °C. Example 2: Dose-response, co-culture of HI-MSC and HSC

Aim

To determine the optimal dose of HI-MSC, used in co-culture with CD34+ hematopoietic stem cells (HSC) from peripheral blood, to support satisfactory expansion of HSC, while retaining their stem cell characteristics.

Summary of setup:

• CD34+ HSC from four different donors and HI-MSC from four different donors.

• Each HSC:HI-MSC donor pairing is unique, for greatest biological variation.

• Four different HI-MSC concentrations are tested and compared to CD34+ HSC monoculture.

Materials and methods

Generation of HI-MSC

See Materials and Methods for Example 1.

Collection of human blood cells

Mobilized peripheral blood from healthy donors was collected from healthy donors after informed consent, as approved by the Central Denmark Region Committees on Health Research Ethics, journal number 1-10-72-144-19.

A standard leukapheresis procedure was applied at the Blood Bank of Aarhus University Hospital, Aarhus, Denmark. HSCs were mobilized through selfadministration of 960 pg G-CSF subcutaneously once daily for four days leading up to harvest day. Donor Medical Evaluation and leukapheresis harvest procedure was performed following the JACIE and WMDA standards. The final leukapheresis product was stored for up to 24h at 4 °C until use.

Isolation of mobilized CD34+ HSC from peripheral blood

CD34+ HSCs were isolated from leukapheresis products by immunomagnetic separation. The CD34+ enrichment was performed on the CliniMACS Prodigy® (Miltenyi Biotec), using the predefined "CD34 enrichment" standard procedure, according to the instructions by the manufacturer. The purity, yield, and viability of the isolated cells were evaluated by flow cytometry. The CD34+ HSC were resuspended in CryoStor CS10 (STEMCELL Technologies), and stored at -140 °C until further use. Dose-response co-culture of HI-MSC and CD34+ HSC

Cryopreserved CD34+ HSC were thawed in a 37 °C water bath and transferred to pre-warmed StemSpan SFEM II followed by centrifugation at 300xg for 5 min. The cells were resuspended in StemSpan SFEM II and the post-thaw absolute concentration of viable CD34+ HSC was determined by flow cytometry. 50.000 viable CD34+ HSC /mL were seeded in 12-well plates to a total volume of 1.5 mL in each well. Cells were cultured in HSC medium, consisting of StemSpan SFEM II supplemented with 30 ng/mL rhSCF (STEMCELL Technologies), 10 ng/mL rhTPO (STEMCELL Technologies), 10 ng/mL rhFLT3L (STEMCELL Technologies), 10 ng/mL rhIL-6 (STEMCELL Technologies), 35 nM UM171 (STEMCELL Technologies), and 20 mg/mL streptomycin and 20 U/mL penicillin. Dose-response co-cultures were prepared by addition of varying concentrations of HI-MSC to the relevant wells. Cell cultures were maintained in a humidified CO2-incubator at 37 °C and 5% CO2 for 6 days after which the viability, CD34+ expansion fold, and stem cell phenotype of the cultured HSC were determined by flow cytometry.

Cellular phenotyping of HSC using flow cytometry

Antibodies, DNA dye and buffers

From BD Biosciences: CD34-PE (clone 581), CD38-BV605 (clone HB7), CD45-FITC (clone 2D1), CD90-PE-Cy (clone: 5E10), CD133-APC (clone: W6B3C1), Brilliant Stain Buffer. From Bio Legend: CD201-APC (Clone: RCR-401). 7AAD was used to identify non-viable cells.

Staining of cells:

HSC were harvested, washed, and resuspended in PBS with 0.1% BSA. For the absolute count of HSCs, cells were stained with 7AAD, CD45 and CD34 antibodies for 15 min at RT. For the cellular phenotyping, cells were stained with 7AAD and antibodies against CD45, CD34, CD90, CD38, CD133, and CD201 antibodies for 30-45 min at RT and washed once with PBS/BSA buffer.

Data acquisition and analysis:

The acquisition of stained cells was performed on a NovoCyte 3000 with a 3-laser configuration (405 nm, 488 nm, 637 nm) (Agilent Technologies), using the NovoExpress Software (versions 1.5.0-1.5.6, Agilent Technologies). Calibration of the cytometer was performed daily, using the NovoCyte QC particles (Agilent Technologies). Compensation was performed monthly, using the compensation beads (anti-Mouse Ig, K, cat: 51-90-9001229 and Negative Control, cat: 51-90- 9001291, BD Biosciences) and 2 uL of each relevant antibody. All gating strategies were based on fluorescence Minus One (FMO) controls. In all cases, the gating strategy involved securing a stable acquisition flow in a time vs. forward scatter (FSC) plot, followed by removal of doublets in a FSC-A vs. FSC-H plot. Unless otherwise stated, the parent population of CD34+ HSC is viable CD45+ cells.

Results

After 6 days of culture, the expansion fold of the CD34+ HSC was determined, defined as the total number of viable CD34+ HSC present after culturing relative to starting number of cells. When compared to the CD34+ HSC monoculture, the co-cultures displayed a dose-dependent increase in the expansion (Figure 3). Overall, approximately 2-3x more CD34+ cells were obtained, when using HI-MSC as expansion support, with a maximum effect at 100.000 HI-MSC/ mL. With the highest concentration at 500.000 HI-MSC/mL, the positive effect seemed to decrease.

The viability of the CD34+ HSC was also consequently higher in all the cocultures, as compared to the CD34+ HSC monoculture (Figure 4). Only CD34+ HSC in the co-cultures could sustain the high viability measured at baseline (DO).

To evaluate the sternness of the cultured CD34+ HSC, the expression of several surface markers, associated with stem cells function, were investigated. Here, a dose-dependent induction was also observed for the total amount of CD34+ HSC expressing the relevant stem cell markers (Figure 5), with a similar tendency to reach a maximal effect at 100.000 HI-MSC/mL after which the effect dropped with higher HI-MSC concentration.

Similar results have also been obtained in assays using HI-MSC that have been stored for even longer than 3-4 days. Cells stored at 4°C at several different time points have been tested, as long as 2 months, 90 days and even 4 months showed capability to support CD34+ expansion (data not shown). At present, the inventors have not been able to establish a time point where after the stored HI- MSC cannot support expansion. Conclusion

The supportive effect of HI-MSC on CD34+ HSC expansion is dose-dependent.

Similarly, the induction or preservation of a relevant stem cell phenotype is also dose-dependent.

Moreover, it was a general observation that the viability of the cultured HSC was always highest in the co-cultures as compared to the HSC monocultures.

The dose of 20.000 HI-MSC/mL was chosen as the optimal dose, as only a subtle advantage was obtained using a 5x higher dose of HI-MSC.

HI-MSC can be stored for sustained periods, whilst preserving their expansion capabilities.

Example 3: Effect of stem cell donor variation

Aim

To investigate if the biological variation of either CD34+ HSC donor or HI-MSC donors has an impact on the degree of supportive effect of HI-MSC on CD34+ HSC proliferation and stem cell characteristics.

Summary of setup:

• CD34+ HSC from four different donors and HI-MSC from four different donors.

• Each CD34+ HSC donor is tested with each of the four HI-MSC donors.

• The optimal HI-MSC dose determined in Example 2 is applied.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Collection of human blood cells

As described in Example 2.

Co-culture of HI-MSC and CD34+ HSC

As described in Example 2, but only using a dose of 20.000 HI-MSC/mL.

Cellular phenotyping using flow cytometry

As described in Example 2.

Results

To determine whether the effect of the biological variation of either HSC donor or HI-MSC donor had the greatest effect on the expansion of CD34+ HSC, a culture matrix of 4 different CD34+ donors and 4 different HI-MSC donors was prepared. There was no overlap between HSC and HI-MSC donors. After 6 days of culture, the expansion fold of the CD34+ HSC in co-cultures with HI-MSC was evaluated and compared to the expansion in the corresponding CD34+ HSC monoculture (Figure 6). The level of HI-MSC induced expansion was relatively similar within each HSC donor, but differed more across HSC donors. The tendency was similar, when looking at the total amount of CD34+ HSC with a relevant stem cell phenotype induced within each HSC donor (Figure 7). On average, approximately 3x more CD34+ HSC expressing the relevant markers were produced in the cocultures compared to monocultures.

Conclusion

The support of HI-MSC on HSC expansion depends to a higher degree on the HSC donor than on the HI-MSC donor variation.

Example 4: Rescue effect over time

Aim

To investigate if HI-MSC exerts a rescue effect on cultured CD34+ HSC, which increases their viability.

Summary of setup:

• Same experimental setup as described in Example 3.

• Viability of CD34+ HSC is measured at baseline (DO), D3 and D6.

Materials and methods

Identical to Example 3, with an additional sampling point on D3 of the culture.

Results

As a consistent observation, the viability of CD34+ HSC is markedly higher in HI- MSC co-cultures compared to HSC monocultures after 6 days of culturing. By measuring the viability on DO, D3, and D6 of the culture, it was possible to identify that HI-MSC seemingly reduces a drop in CD34+ HSC viability, as measured in D3 (Figure 8). A similar effect was observed for all four CD34+ HSC donors included. Conclusion

HI-MSC has a protective- or rescue effect on cellular viability of cultured CD34+

HSC.

Example 5: HI-MSC compared to the proprietary small molecule UM171

Aim

To investigate whether HI-MSC can serve as a substitute for a small molecule, UM171, which is widely used for CD34+ HSC expansion ex vivo.

Summary of setup:

• Same experimental setup as described in Example 3, but only using one HCS donor and one HI-MSC batch.

• UM171 and cytokines are used in HSC monoculture and in a co-culture, and compared to a co-culture without UM171, but still with cytokines.

Materials and methods

Generation of HI-MSC

As described in Example 1.

HI-MSCs are here stored in saline/2% HSA.

Collection of human blood cells

As described in Example 2.

Co-culture of HI-MSC and CD34+ HSC

As described in Example 2, but only using a dose of 20.000 HI-MSC/mL.

In the indicated cultures, no UM171 was added to the HSC medium.

Cellular phenotyping using flow cytometry

As described in Example 2.

Results

To facilitate clinical translation of the developed CD34+ HSC expansion protocol, it is essential to identify reagents and processes, which are compatible with current Good Manufacturing Practice (GMP). The proprietary small molecule UM171 patented for clinical use by ExCellThera, is applicable for use in HSC expansion.

We hypothesized that the observed supportive effect of HI-MSC on HSC expansion could be obtained without the presence of UM171. As seen in Figure 9, the expansion fold of viable CD34+ HSC was similar in the co-cultures, regardless of the presence of UM171 in the HSC medium. Moreover, the viability of the cultured HSC improved in the co-cultures compared to the baseline viability (Figure 10), and was also higher than in the HSC monoculture.

The sternness of the generated CD34+ HSC was evaluated by an associated stem cell phenotype. Comparing the co-cultures, the number of CD34+ CD133+ HSC was somewhat affected by the omission of UM171, yielding similar levels as the HSC monoculture (Figure 11). Still, there was a 7,5x increase in the absolute number of CD34+ CD133+ HSC in the co-culture without UM171, compared to baseline (monoculture vs. baseline: 6x; co-culture + UM171 vs. baseline 12x). The expression of CD201 was completely absent, when UM171 was removed from the HSC medium. From several published studies, it has been shown that CD201 expression is induced by UM171, thus confirming the observations from the current study. Finally, the number of CD34+ CD90+ and CD34+ CD38- HSC produced in the co-cultures was slightly lower, when UM171 was omitted.

Nevertheless, 2x more CD34+ CD90+ HSC was produced compared to HSC monoculture and 132x more than at baseline. A slightly higher, yet similar trend was observed for the amount of CD34+ CD38- HSC.

Conclusion

HI-MSC supports similar expansion fold and viability of cultured CD34+ HSC both in the presence and absence of UM171 in the HSC medium. A slightly different stem cell phenotype arises, when UM171 is not applied in co-culture, yet substantial amounts of CD34+ HSC with a relevant phenotype is still produced. This has the potential to pave the way for new clinically relevant expansion protocols for CD34+ HSC, which eliminates the need of UM171 for clinical use.

Example 6: Supportive effect of HI-MSC derivatives on HSC expansion

Aim

To investigate whether the observed supportive effect of HI-MSC is contactdependent or -independent and moreover, if HI-MSC-primed HSC medium can exert the same effect as the HI-MSC.

Summary of setup:

• Same experimental setup for mono- and contact-dependent co-culture, as described in Example 3. • The entire setup is repeated three times, using three different CD34+ HSC donors each matched with a different HI-MSC batch.

• Primed HSC medium is prepared with both new HI-MSC (3-4 days post-HI) and old (3-5 weeks post-HI), using HI-MSC from the same donor that have merely been heat-inactivated at different time-points. The priming is performed for 24h and the final concentration of the applied primed HSC medium corresponds to the same dose of HI-MSC used in the co-cultures (20.000 HI-MSC/mL).

• In one setup, a highly concentrated HI-MSC primed HSC medium is prepared, to investigate if a similar supportive effect can be obtained from a stronger "brew", which is, nonetheless, diluted to the same final concentration as the otherwise applied primed medium.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Collection of human blood cells

As described in Example 2.

Generation of HI-MSC primed HSC medium

One day before co-culture setup, primed HSC medium was prepared. Initially, the 7AAD staining and concentration of HI-MSCs were evaluated by flow cytometry. Subsequently, HI-MSCs were diluted to a concentration of 60.000 HI-MSC/mL, using StemSpan SFEM II, and transferred to a T25 culture flask. The HI-MSC suspension was stored in the CO2-incubator at 37°C and 5% CO2 for 24 hours. Next day, the cell suspension was centrifuged at 440 x g, and the supernatant was collected to be used in the culture setup as HI-MSC-primed HSC medium.

In one setup, priming of the HSC medium was performed with 2x10^6 HI- MSC/mL, but otherwise as described above.

Co-culture of HI-MSC and CD34+ HSC

HSC monocultures: Three different monocultures were prepared for each culture setup, all with the same CD34+ HSC seeding density and culture medium supplements as described in Example 2. One monoculture was prepared exactly as described in Example 2. A second monoculture was set up with StemSpan SFEMII medium, which was pre-incubated without supplements for 24h in a CO2 incubator prior to culture setup, to serve as a control for the third monoculture setup. This last CD34+ HSC monoculture setup was prepared with the HI-MSC primed HSC medium. Here, 0,5 mL of primed HSC medium was added to each relevant well in a 12-well plate. The final volume in the well was 1,5 mL, thus diluting the primed HSC medium corresponding to a final concentration of 20.000 HI-MSC/mL, to match the concentration of HI-MSC used in the contact-dependent setup. In one setup, 15 uL of the concentrated, HI-MSC-primed HSC medium was used, again diluting the primed HSC medium corresponding to a final concentration of 20.000 HI-MSC/mL.

Contact-dependent co-cultures-. As described in Example 2, but only using a dose of 20.000 HI-MSC/mL.

Contact independent co-cultures: A cell culture insert with a PET membrane of pore size of 0.4 pm (Falcon) separated CD34+ HSC and HI-MSC. Initially, 1,5 mL of CD34+ HSC cell suspension was transferred to a 12-well plate after which the culture insert was placed in the well. 0,5 mL of HI-MSC suspension was placed in the culture insert, making the total volume of the setup 2 mL. 50.000 viable CD34+ HSC /mL and 20.000 HI-MSC were used, to match the condition of the contact-dependent co-culture. At day 6 of the culture, the insert was removed from the well, allowing harvest of the cultured CD34+ HSC.

Cellular phenotyping using flow cytometry

As described in Example 2.

In vitro CFU assay for functional evaluation of cultured CD34+

For the colony-forming unit (CFU) assay, the StemMACS™ HSC-CFU Assay Kit (Miltenyi Biotec) was applied. The assay was performed according to the manufacturer's instructions. Briefly, the total number of CD34+ cells was determined by flow cytometry, as described above, and the cell concentration was adjusted to 250 CD34+ cells/mL with StemSpan SFEM II. Cells were seeded in a 96 round-bottom well plate with an average of 2,5 cells/well and the assay medium was added. Plates were placed within a humidity chamber in a CO2- incubator (37 °C and 5% CO2). After 14 days, cells were stained with an antibody cocktail, containing antibodies against CD14, CD15, and CD253a (glycophorin A). After 10-15 min of incubation, cells were acquired on a NovoCyte Quanteon (Agilent Technologies) with a 4 laser configuration (405 nm, 488 nm, 561 nm, 637 nm), using the NovoExpress Software (version 1.5.6). A minimal number of 250 events/well was required, of which at least 35 events needed to be positive for either of the three cell surface markers. The gating strategy was performed as advised by the manufacturer and consisted in identifying a BFU-E colony, in which only CD235a is expressed, making it possible to adjust gates for CD14 and CD15. Based on the percentwise expression of the three surface markers the distribution of different colony types was determined for each well (Table 1).

Table 1. Surface marker evaluation for determination of colony types. The five different colony types are determined based on the marker distribution pattern indicated in the table.

Results

To further elaborate on the possible mechanisms behind the supportive effect of HI-MSC on CD34+ HSC expansion, we investigated if the effect was contactdependent or even required the presence of HI-MSC, using a HI-MSC primed HSC medium. Initially looking at the cellular proliferation, the expansion fold of CD34+ HSC was significantly higher in the contact-dependent co-culture, as compared to the HSC monoculture, after 6 day of culture (Figure 12). This correlates with previous findings from other co-culture studies (Figure 3, 6, 9). Moreover, the contact-independent co-culture yielded a slightly higher, yet comparable, expansion fold as the contact-dependent counterpart. This indicates that the expansion support of HI-MSC on cultured CD34+ HSC is not contact-dependent. However more surprisingly, similar expansion support could be obtained using HSC medium primed for 24h with HI-MSC. This effect could not be ascribed to the HSC medium alone, as pre-incubation of this for 24h prior to culture setup, did not affect the observed CD34+ expansion fold in the HSC monoculture (Figure 12, monoculture, pre-inc.med.). This consequently points to an effect of HI-MSC derivatives present in the HI-MSC primed HSC medium that are capable of exerting a similar supportive effect as the HI-MSC. This effect could also be obtained with HI-MSC that had been stored for several weeks at 4 C°, which could suggest that the supportive effect of HI-MSC is sustained for longer periods of time.

The observed viability was highest in the cultures containing HI-MSC or their derivatives (Figure 13). The support of higher viability in co-cultures over HSC monocultures is a consistent observation across several other experiments (Figure 4, 8, 10).

In addition to prominent viability, the total amount of CD34+ HSC expressing relevant stem cell markers was also markedly higher in the cell cultures containing HI-MSC or HI-MSC derivatives as compared to HSC monocultures with no HI-MSC components (Figure 14). Similar to the CD34+ expansion fold, the HI- MSC induced CD34+ stem cell phenotype does not require physical contact or even direct presence of HI-MSC.

To extend on the applicability of the HI-MSC primed medium as a culture supplement, a priming was set up, using 2x10^6 HI-MSC/mL in HSC medium. This more concentrated HI-MSC primed HSC medium was subsequently diluted in the CD34+ HSC culture, to match the use of a final concentration of 20.000 HI- MSC/mL. Compared to the HI-MSC primed HSC medium, which was primed with a smaller concentration of HI-MSC, a similar CD34+ expansion fold (Figure 15 (A)) and CD34+ stem cell phenotype (Figure 15 (B)) was produced.

The sternness of the CD34+ HSC was further evaluated by investigating the differentiation potential of the cultured CD34+ HSC, a functional in vitro colony forming unit (CFU) assay was performed. This assay supports differentiation of the myeloid progenitor cells and not the lymphoid. Nevertheless, it estimates the presence of five different colony types; Burst-forming unit-erythroid (BFU-E), CFU-granulocyte (G), CFU-granulocyte macrophage (GM), CFU-macrophage (M), and CFU-granulocyte erythrocyte macrophage megakaryocyte (GEMM). Compared to non-expanded cells, both the total number of colonies and the colony types changed markedly after 6 days of expansion in either monoculture or co-culture. The total number of colonies per plate was highest for the non-expanded CD34+ HSC, with 92 colonies/plate (average of three plates), while the number was reduced to 50% of this, yielding an average of 46 colonies/plate for the cultured cells (average of 1 plate per cultured cells)(Figure 16 (A)). The colony distribution shifted after expansion, after which the fraction of BFU-E dropped particularly, while no CFU-GEMM could be identified among the cultured CD34+ HSC (Figure 16(B)). However, both the reduction of the number of colonies and a changed colony distribution pattern correlate with previously obtained results (data not shown). The results from this study points to preservation of the in vitro myeloid differentiation potential of the cultured cells.

Conclusion

HI-MSC and HI-MSC primed HSC medium supports higher CD34+ expansion fold and production of more CD34+ HSC with a relevant stem cell phenotype than HSC monocultures without HI-MSC components. Moreover, the HI-MSC induced effect in co-cultures is seemingly not contact-dependent. The expanded CD34+ HSC preserve a differentiation potential, showing that functionally competent HSC are produced after expansion. The HI-MSC primed HSC medium contains a vesicular component, which may contribute to the supportive effect of the primed HSC medium. Moreover, HI-MSC that have been stored for several weeks at 4 C° can be used for priming of HSC medium and support similar expansion and CD34+ phenotype as using freshly prepared HI-MSC for the medium priming. Combined with the observation that a highly concentrated HI-MSC priming of HSC medium can be performed, HI-MSC or perhaps even primed medium/solution could be offered as a cell culture supplement.

Example 7: Stability of HI-MSC when used as expansion support

Aim

To test if HI-MSC are stable for 6 days of culture with or without the presence of CD34+ HSC.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Cell counts and viability measurements

As described in Example 1.

Stability testing of HI-MSC

20.000 HI/mL was seeded in StemSpan SFEMII medium in a 12-well plate, using a final volume of 1,5 mL in each well. The plate was placed in a humidified CO2- incubator at 37 °C and 5% CO2. HI-MSC counts and 7AAD staining was evaluated after 6 days of incubation.

Collection of human blood cells

As described in Example 2.

Co-culture of HI-MSC and CD34+ HSC

As described in Example 2, but only using a dose of 20.000 HI-MSC/mL.

Results

Adding to the storage stability at 4 C°, we tested the stability of HI-MSC in a cell culture setting. After 6 days on incubation, the concentration of HI-MSC remained stable compared to the starting concentration of 20.000 HI/mL, both when seeded as a HI-MSC monoculture (Figure 17(A)) and in co-culture with CD34+ HSC (Figure 17(B)).

Conclusion

HI-MSC remain stable in cell culture medium for several days, both in HI-MSC monoculture and when used in co-culture with CD34+ HSC.

Example 8: Support of CD34+ expansion in mono- and co-culture in a clinically relevant growth medium

Aim

To test whether the expansion CD34+ mono- and co-culture can be supported in a HSC growth medium, which is compatible with clinical applications, i.e. GMP compliant.

Summary of setup:

• The basal HSC growth medium, StemSpan SFEMII is replaced with a clinically compatible version, StemSpan AOF.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Cell counts and viability measurements

As described in Example 1. Collection of human blood cells

As described in Example 2.

Co-culture of HI-MSC and CD34+ HSC

As described in Example 2, but only using a dose of 20.000 HI-MSC/mL.

The basal growth medium is replaced in all steps with StemSpan AOF (STEMCELL Technologies). All cytokines were from PeproTech.

Cellular phenotyping using flow cytometry

As described in Example 2.

Results

After 6 days of culture, the expansion fold of the CD34+ HSC was determined, defined as the total number of viable CD34+ HSC present after culturing relative to starting number of cells. When using StemSpan AOF as basal HSC growth medium, the expansion fold of CD34+ cells in culture is highest in co-culture relative to the HSC monoculture (Figure 18A). In the co-culture, approximately 50% more cells are obtained after 6 days of expansion, compared to the matching monoculture. The viability after 6 days was 95% and 97% in mono- and coculture, respectively (data not shown). Moreover, CD34+ cells with a relevant phenotype was produced in both mono-and co-culture (Figure 18B), with the highest amount of cells produced in the co-culture.

Conclusion

The expansion of CD34+ HSC in monoculture and co-culture is supported, when using StemSpan AOF as the basal HSC growth medium. Similar CD34+ expansion folds are observed after 6 days of culture for both StemSpan SFEMII (Figure 9,12) and StemSpan AOF (Figure 18A). Furthermore, more CD34+ cells with a relevant phenotype are produced in the co-culture, as compared to the monoculture, when using StemSpan AOF as HSC growth medium, and similar results are also observed, when using StemSpan SFEMII (Figure 5,7,11,14). Consequently, it is feasible to expand CD34+ HSC in mono- and co-culture in several types of HSC growth medium. Example 9: Viability, metabolic activity, and cellular morphology of MSCs after inactivation with different methods

Aim

To test how viability, metabolic activity, and cellular morphology of MSC is affected after inactivation with either heat or irradiation.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Additional measurements of apoptosis levels and metabolic activity was added to the protocol. These are described in more detail below.

Generation of irradiated MSC

Freshly harvested MSC were centrifuged at 440xg for 5 min. and resuspended in 20 mL saline + 2% HSA to reach a concentration of 3x10^6 cells/mL. 5 mL of the cell suspension was added to a sterile 50 mL tube and irradiated (14 Gy). Cell concentration, viability, apoptosis, metabolic activity, and plastic adherence were evaluated immediately after irradiation. 5x10^5 cells were seeded in T75 culture flasks for evaluation on day 1 and day 3 post-irradiation.

Plastic adherence

MSC (control), HI-MSC, and irradiated MSC were seeded immediately postinactivation at a concentration of 2x10^4 cells/mL in MSC medium (see Example 1) in T25 flasks, in a total volume of 5 mL, to test for plastic adherence. Culture flasks were placed in a humidified CO2-incubator at 37°C and 5% CO2. On day 1 and 3 after inactivation, the culture flasks were inspected by light microscopy, to evaluate adherence and cellular morphology.

Harvest of cells

On day 1 and 3, the seeded cells were harvested to facilitate evaluation of viability, metabolic activity, and apoptosis levels. The irradiated cells were harvested as described in Example 1, Cultivation of MSC. HI-MSCs were stored at 4°C in saline + 2% HSA in cryotubes, containing 1 mL of 5x10^6 cells/mL. HI- MSC were sampled directly from the tube and used in the analyses.

Cell counts and viability measurements

As described in Example 1. This analysis was also performed with irradiated MSC.

Apoptosis assay

Apoptosis levels were evaluated using Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & propidium-iodide (PI) (Thermo Fisher Scientific). The assay was performed according to the instructions by the manufacturer. In detail, 1x10^5 cells were transferred to an 1.5 mL tube, washed with PBS + 0.1% BSA, and centrifuged at 440xg for 5 min. After removing the supernatant, the pellet was resuspended in 100 pL lxAnnexin-binding buffer (to reach a concentration of 1x10^6 cells/mL). The 100 pL cell suspension was transferred to a tube, suitable for flow cytometry, containing 1 pL PI (100 pg/mL) and 5 pL Alexa Fluor 488 Annexin V and incubated in darkness for 15 min. Following incubation, 400 pL lxAnnexin-binding buffer was added, and the sample was analyzed by flow cytometry. The gating strategy was performed as advised by the manufacturer, identifying live cells as Annexin V- PT, apoptotic cells as Annexin V⁺ PT, and dead cells as Annexin V⁺ PI⁺.

Metabolic activity assay (XTT assay)

Using CyQUANT XTT Cell Viability assay (Thermo Fischer Scientific), the metabolic activity of HI-MSC and Irradiated MSC were investigated. XTT reagent and electron coupling reagent were thawed according to the instructions by the manufacturer. HI-MSC and irradiated MSC were added to a 96-well plate in triplicates, with each well containing 5x10^4 cells. XTT working solution was prepared by adding 6 mL XTT reagent and 1 mL electron coupling reagent. Working solution was used immediately by adding 70 pL to each of the wells containing cells, followed by 2 hours of incubation in a humidified CO2 incubator. Medium controls without cells were included in this analysis. After 2 hours of incubation, the absorbance was analyzed with an absorbance plate reader at 450 nm.

Results

To investigate if heat inactivation of MSC has a different impact on cellular characteristics compared to other inactivation methods, selected features of HI- MSC and irradiated MSC were compared. Plastic adherence and spindle shaped morphology was observed for the irradiated MSC at both DI (24H) and D3 (72H) post-inactivation. As expected, HI-MSC did not adhere and retained a spherical morphology (Figure 19). The viability of the cells was evaluated using two different assays. Staining cells with 7AAD demonstrated distinctive levels of 7AAD- cells (viable cells) over time for both types of inactivated MSC (Figure 20A). 72H post-inactivation, the viability remained prominent for the irradiated cells, while only low levels of 7AAD- were detected for HI-MSC. The latter observation correlates with previous results (Figure 1). Moreover, the fraction of both apoptotic (Annexiv V+ PI-, Figure 20B) and necrotic cells (Annexiv V+ PI+) only slightly increased for irradiated MSC over time, while necrotic cells constituted the main fraction of HI-MSC 72H after inactivation. Evaluation of metabolic activity demonstrated a retained activity for irradiated MSC (Figure 21), which was comparable to non-inactivated MSC. Conversely, the signal measured for HI-MSC was similar to the background signal/ blank instantly after inactivation. As a final note, using irradiated MSC in co-culture with CD34+ HSC also supported the expansion of the latter (data not shown). Adherent irradiated MSC could still be observed in the co-culture wells after 6 days of incubation. HI- MSC did not adhere and maintained to be present in the culture medium.

Conclusion

Inactivation of MSC with irradiation creates cells that maintain several features of non-inactivated/control MSC, namely plastic adherence, spindle-shaped morphology, and metabolic activity. In contrast, heat-inactivation of MSC produces cells which have lost the ability to adhere to plastic, have a different morphology, and have no metabolic activity, when compared to their noninactivated counterpart. Collectively, this demonstrates that heat inactivation yield cells with significantly different features from those obtained with inactivation by irradiation.

Example 10: Primed medium from HI-MSC stored at -80 °C prior to usage maintain expansion support on HSC

Aim

To test if storage of HI-MSC primed HSC medium at -80 °C affects its supportive effect on CD34+ HSC expansion.

Materials and methods

Generation of HI-MSC

As described in Example 1.

Collection of human blood cells

As described in Example 2.

Generation of primed medium from HI-MSC

As described in Example 6, but using StemSpan AOF. Primed medium was either used immediately after generation or stored at -80 °C for 9 days before usage.

Co-culture of HSC and HI-MSC

Co-cultures of CD34+ HSC and HI-MSC were performed as described in Example 2, but only using a dose of 20.000 HI-MSC/mL. In addition, all cytokines were changed to GMP-compliant versions from Miltenyi Biotec and the base medium was the clinically compatible version, StemSpan AOF, described in Example 8. Cells were seeded in a 24 well plate, with a total volume of 1 mL in each well.

Cellular phenotyping of HSC using flow cytometry

As described in Example 2.

Results

To elaborate on the usability of the HI-MSC primed HSC medium, we tested if storage at -80 °C would affect the ability to support CD34+ expansion. The proliferation of CD34+ HSC was supported by both freshly prepared and prefrozen HI-MSC primed HSC medium (Figure 22A) and a comparable level of expansion was observed in co-culture with HI-MSC. Moreover, all cultures containing HI-MSC or HI-MSC derivatives performed superior to HSC monoculture, which correlates with previous findings (Figure 12).

The viability of the cultured CD34+ HSC was also consistently higher in all cultures containing MSC or their derivatives (Figure 22B). This has been a repeated observation across several experiments (Figures 4, 8, 10, 13). When evaluating the total amount of CD34+ HSC expressing relevant stem cell markers, this was markedly higher in the co-culture and monocultures with HI- MSC derivatives, compared to the monoculture without (Figure 23). This matches previous findings (Figure 14). Interestingly, the pre-frozen HI-MSC primed medium also induced a relevant stem cell phenotype in the CD34+ HSC.

Conclusion

Similar supportive effect on HSC expansion can be obtained with pre-frozen (-80 °C) and freshly prepared HI-MSC primed HSC medium. This translates to the possibility of having ready-to-use preparations of a HI-MSC primed solution, instead of having to prepare it fresh before use. Example 11. Effect of long-term storage at 4°C on stability and expansion support capability of HI-MSC.

Aim

To test if HI-MSCs are stable after long-term storage at 4°C and maintain their expansion support capabilities.

Materials and methods

Generation of HI-MSC

As described in Example 1. The HI-MSC evaluated here has been stored in StemSpan SFEMII with an addition of 8,4 pL pen/strep per mL of HI-MSC solution, giving a final pen/strep concentration of 20 U/mL.

Collection of human blood cells

As described in Example 2.

Co-culture of HSC and HI-MSC

As described in Example 10.

Results

To test the impact of long-term storage on HI-MSC stability, the concentration and 7AAD staining was tested 17 and 21 months after inactivation. The concentration of HI-MSC remained stable at 4°C after long-term storage and was comparable to the concentration measured immediately after inactivation (Figure 24A). A similar tendency was observed for the level of 7AAD staining (Figure 24B), indicating that they retain a permeable, yet stable cell membrane over time.

Used in co-culture with CD34+ HSC, the long-term stored HI-MSC (21 months) provided higher expansion fold and increased viability, compared to HSC monoculture (Figure 25 A and B), and additionally supported a relevant stem cell phenotype in the cultured HSC (Figure 25C). This correlates with previous findings, in which HI-MSC stored for several weeks at 4 C° also provided favorable culture conditions to CD34+ HSC (Figure 13). In addition, the HI-MSC used in coculture after 21 months of storage did not adhere to the culture plastic, as evaluated by microscopy, thus preserving the non-adherent features previously demonstrated (Figure 19). Collectively, this indicates that HI-MSC can be stored for several months without losing their ability to support HSC expansion. Conclusion

HI-MSCs remain stable in terms of concentration and viability staining after longterm storage at 4°C. In addition, long-term stored HI-MSCs maintain their supportive capacity of CD34+ HSC expansion.

References

1. Kadereit S, Deeds LS, Haynesworth SE, Koc ON, Kozik MM, Szekely E, et al. Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer. Stem Cells. 2002;20(6):573-82.

2. Zaker F, Nasiri N, Oodi A, Amirizadeh N. Evaluation of umbilical cord blood CD34 (+) hematopoietic stem cell expansion in co-culture with bone marrow mesenchymal stem cells in the presence of TEPA. Hematology. 2013;18(l):39-45.

3. Flores-Guzman P, Flores-Figueroa E, Montesinos JJ, Martinez-Jaramillo G, Fernandez-Sanchez V, Valencia-Plata I, et al. Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood.

Cytotherapy. 2009; ll(7):886-96.

4. Yamaguchi M, Hirayama F, Kanai M, Sato N, Fukazawa K, Yamashita K, et al. Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells. Exp Hematol. 2001;29(2): 174-82.

Items

1. A process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs. 2. The process according to item 1, wherein the time sufficient to expand the HSCs is at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.

3. The process according to item 1 or item 2, wherein the HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell co-culture, such as a non-contacting co-culture or a contacting co-culture.

4. The process according to item 3, wherein the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.

5. The process according to item 1, wherein the HI-MSC cells are placed in spatial contact with the HSCs.

6. The process according to any of the preceding items, wherein at least 1000 HI-MSC cells are provided, such as at least 5000, such as at least 10,000, such as at least 20,000 HI-MSC cells are provided, such as even up to 6*10⁶ HI- MSCs, such as even 12*10⁶ HI-MSC cells are provided.

7. The process according to any of the preceding items, wherein between 40.000/cm² and 70.000/cm² HI-MSC cells are provided, such as 50.000/cm², such as even 60.000/cm² HI-MSC cells are provided, preferably 60.000/cm² HI- MSC cells are provided.

8. The process according to any of the preceding items, wherein at least 1000 HSCs are provided, such as at least 5000, such as at least 10,000, such as at least 20,000, such as at least 30,000, such as at least 40,000, such as at least 50,000 HSCs are provided.

9. The process according to any of the preceding items, wherein at least 5% HI- MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b. 10. The process according to any of the preceding items, wherein the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood.

11. The process according to any of the preceding items, wherein the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.

12. The process according to item 10, wherein the adipose derived MSCs are derived from a stromal vascular fraction (SVF).

13. The process according to any of the preceding items, wherein the HSCs have been genetically modified prior to, during and/or after expansion, such as by the use of CRISPR-Cas9.

14. The process according to any of the preceding items, wherein the HI-MSC cells are provided directly after heat inactivation, or the HI-MSC cells have been stored at e.g. below 10°C such as below 5°C, such as in the range 10°C-0.1°C, between -24°C and 5°C, or have been stored at -80°C, such as having been cryogenically stored.

15. The process according to any of the preceding items, wherein the HI-MSC cells have been stored at 4 °C and used 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation.

16. The process according to any of the preceding items, wherein the HI-MSC and/or the HSC medium is free from chemical cell inactivation agents, such as mitomycin C. 17. The process according to any of the preceding items, wherein the HI-MSC cells are metabolically inactivated.

18. The process according to any of the preceding items, wherein the provided HI- MSCs in step a) has maintained their cellular integrity, such as maintained their cellular integrity

- for at least 15 days, such as at least 30 days or such as at least 60 days, when stored in a temperature range between 0.1 and 10°C, such as 1- 8°C, such as 2-6°C, such as 3-5°C, or such as around 4°C; and/or

- for at least 2 days, such as at least 7 days, such as at least 15 days, such as at least 30 days, such as at least 60 days, when stored in a temperature range 11-28°C, such as 15-28°C, or such as 18-25°C.

19. The process according to any of the preceding items, wherein the HSC growth media comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.

20. A process of providing a pre-conditioned media according to item 1, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

21. A process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

22. The process according to item 20 or 21, wherein the media is selected from

- a growth media, such as a HSC growth media; and

- a liquid cell medium, such as a balanced salt solution (BSS), such as PBS, preferably isotonic saline and/or a pharmaceutical acceptable composition.

23. The process according to any of items 20-22, wherein the HSC growth media is supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.

24. The process according to any of items 20-23, wherein at least 60.000 HI- MSC/mL medium is added, such as up to 2x10^6 HI-MSC/mL medium, preferably 2x10^6 HI-MSC/mL medium.

25. The process according to any of items 22-24, further comprising supplying a container comprising HSCs with an amount of the pre-conditioned media, sufficient to expand the HSCs.

26. A pre-conditioned media obtained by or obtainable by the process according to any of items 22-24.

27. A container comprising the pre-conditioned media according to item 26.

28. Use of HI-MSCs, such as stored HI-MSCs, as feeder cells for expanding HSCs.

29. Use of HI-MSCs, such as stored HI-MSCs, as cells for pre-conditioning HSC growth media. 30. Use of the pre-conditioned media according to item 26, to expand a population of HSCs.

31. A HSC population of cells obtained by or obtainable by a process according to any of the preceding items 1-19 or 25.

32. The HSC population of cells according to item 31, wherein the cells are positive for the markers CD45, CD34 and

• negative for CD38.

33. The HSC population of cells according to item 31, wherein the cells are positive for the markers CD45, CD34, and

• positive for at least one of the markers selected from the group CD90, and CD133, and/or

• negative for CD38 and CD201.

34. The HSC population of cells according to any of items 31-33, wherein the HSC population of cells has an increased viability, when compared to a HSC monoculture.

35. A container comprising the HSC population of cells according to any of items 31-34.

36. A kit comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.

37. A kit comprising the pre-conditioned media of item 26, and optionally comprising instructions for expanding HSCs.

38. The HSC population of cells according to any of items 31-34, for use as a medicament. 39. The HSC population of cells according to any of items 31-34, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease.

40. The HSC population of cells for use according to item 39, wherein the leukemia is a leukemia selected from the group consisting of Multiple myeloma (MM), Myelodysplastic syndrome (MDS), Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia

(CLL), Chronic myeloid leukemia (CML), Hodgkin lymphoma, and Non-Hodgkin lymphoma (NHL), including : Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma, Burkitt lymphoma, and T-cell lymphoma.

41. The HSC population of cells for use according to item 39, wherein the blood disorder is a blood disorder selected from the group consisting of sickle cell disease, thalassemia, aplastic anemia, and Fanconi anemia.

42. The HSC population of cells for use according to item 39, wherein the immune system disease is an immune system disease selected from the group consisting of severe combined immunodeficiencies (SCID), chronic granulomatous disease (CGD), Wiskott-Aldrich syndrome (WAS), hemophagocytic lymphohistiocytosis (HLH), severe autoimmune diseases, and selected inherited metabolic disorders.

43. A method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease in a patient in need thereof, the method comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.

44. The process according to any of the preceding items, wherein the MSCs are derived from a mammal, and preferably a human being.

45. The process according to any of the preceding items, wherein the MSCs are expanded MSCs. Clauses

1. A process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), such as at least 1000 HI-MSC cells, such as at least 5000, such as at least 20,000 HI-MSC cells, or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs, such as at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.

2. The process according to clause 1, wherein the HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell coculture, such as a non-contacting co-culture or a contacting co-culture, optionally wherein the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.

3. The process according to clause 1, wherein the HI-MSC cells are placed in spatial contact with the HSCs.

4. The process according to any of the preceding clauses, wherein at least 5% HI-MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b.

5. The process according to any of the preceding clauses, wherein the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood, or wherein the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.

6. The process according to any of the preceding clauses, wherein the HI-MSC cells have been stored at 4 °C and used 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation.

7. The process according to any of the preceding clauses, wherein the HSC growth media, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF, comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin.

8. A process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell, such as 60.000 HI-MSC/mL medium, such as up to 2x10^6 HI-MSC/mL medium, preferably 2x10^6 HI-MSC/mL medium; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

9. The process according to clause 8, wherein the HSC growth media, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF, is supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin. 10. A pre-conditioned media obtained by or obtainable by the process according to clause 8 or clause 9.

11. Use of the pre-conditioned media according to clause 10, to expand a population of HSCs.

12. Use of HI-MSCs, such as stored HI-MSCs stored at 4 °C and used 2 days post in-activation, as feeder cells for expanding HSCs.

13. Use of HI-MSCs, such as stored HI-MSCs stored at 4 °C and used 2 days post in-activation, as cells for pre-conditioning HSC growth media.

14. A HSC population of cells obtained by or obtainable by a process according to any of the preceding clauses 1-7, optionally wherein the HSC population of cells are positive for the markers CD45, CD34 and

• positive for at least one of the markers selected from the group CD90, CD133, and CD201; and/or negative for CD38; or

• positive for at least one of the markers selected from the group CD90, and CD133, and/or negative for CD38 and CD201.

15. The HSC population of cells according to clause 14, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease.

Claims

1. A process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells) that are non-viable and metabolically inactive, such as at least 1000 HI-MSC cells, such as at least 5000, such as at least 20,000 HI-MSC cells, or a HI-MSC pre-conditioned HSC growth medium, wherein the preconditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs, such as at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.

2. The process according to claim 1, wherein the HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell co-culture, such as a non-contacting co-culture or a contacting co-culture, optionally wherein the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.

3. The process according to claim 1, wherein the HI-MSC cells are placed in spatial contact with the HSCs.

4. The process according to any of the preceding claims, wherein at least 5% HI-MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b.

5. The process according to any of the preceding claims, wherein the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood, or wherein the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.

6. The process according to any of the preceding claims, wherein the HI-MSC cells have been stored at 4 °C and used 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation, such as the cells have been stored for at least 17 months, at least 21 months.

7. The process according to any of the preceding claims, wherein the HI-MSC cells have been stored below 10°C post in-activation for at least 2 days, such as at least 15 days, such as at least 4 months, such as even 1-2 years.

8. The process according to any of the preceding claims, wherein the HSC growth media, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF, comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin.

9. A process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell that is non-viable and metabolically inactive, such as 60.000 HI-MSC/mL medium, such as up to 2x10^6 HI-MSC/mL medium, preferably 2x10^6 HI- MSC/mL medium; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.

10. The process according to claim 9, wherein the media is a HSC growth media, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF, and supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin and/or penicillin.

11. Use of HI-MSCs that are non-viable and metabolically inactive, such as stored HI-MSCs stored below 10°C for at least 2 days post in-activation, as feeder cells for expanding HSCs.

12. Use of HI-MSCs that are non-viable and metabolically inactive, such as stored HI-MSCs stored below 10°C for at least 2 days post in-activation, as cells for preconditioning HSC growth media.