Patent Application
20190285614
Not applicable.
This application is in the field of substances that promote neurogenesis and gliogenesis, and assays for such substances.
Mesenchymal stromal cells contain a population of multipotent cells, known as mesenchymal stem cells (reviewed in [1]). A major source of mesenchymal stem cells (MSC) in adult mammals is the bone marrow; multipotent cells obtained from bone marrow are known variously as mesenchymal stem cells (MSC), marrow adherent stromal cells (MASC), marrow adherent stem cells, and bone marrow stromal cells (BMSC). Mesenchymal stem cells have been studied as a potential cellular therapy for the repair of neural tissue (reviewed in [2]). Transplantation of MSC or MSC derivatives into the nervous system has been shown to be beneficial in many models of neurodegenerative diseases including stroke, Parkinson's disease, spinal cord injury, multiple sclerosis, and neonatal hypoxic-ischemic brain injury [3-9].
Current evidence suggests that the transplantation of MSC or their derivatives activates endogenous regeneration mechanisms both in injured neural tissue [9-13] and in normal brain tissue [14]. These regenerative processes include enhanced proliferation of endogenous neural stem cells, increased survival of newborn neurons [10-11], gliogenesis [7], and modulation of inflammatory cytokine production [15]. It is thought that the neuroprotection and enhancement of neural proliferation are mediated, at least in part, by diffusible neurotrophic factors and cytokines secreted by the transplanted cells. Indeed, MSC have been shown to secrete a number of growth factors in culture [16, 17]; and the identity of the growth factors secreted can be modulated by transplantation in a neurodegenerative environment [18, 19].
It is thus important to identify the factors produced by MSC, and their derivatives, that are responsible for the neuropoietic and gliogenic activities of mesenchymal cells. The study of interactions between MSC and neural cells in vitro poses the challenge of creating culture conditions that are suitable for several different cell types (e.g., neurons, glial cells, neural stem cells, mesenchymal cells), each having different requirements for substrate and growth media. Indeed, in most systems, co-culture conditions (for example, the presence or absence of serum, or the use of MSC monolayers as substrate for small numbers of neural cells) selectively favor certain cells at the expense of others, which leads to inconsistent results [23-26] and prevents the adequate quantification of MSC effects. For example, certain culture systems are favorable to the growth of neurons, but not of glial cells; and no system has been found that supports the growth of neural precursor cells and the three major types of neural cell (neuron, oligodendrocyte and astrocyte) simultaneously.
The effects of MSC and other substances on proliferation and differentiation of neural stem cells into various neural lineages (i.e., neuropoiesis) are commonly studied in vitro using mitogen-driven neurospheres as a source of neural stem/early precursor cells; subsequently, their differentiation is induced by plating neurospheres on an adhesive substrate and withdrawing the mitogenic growth factors [23-26]. However, cells in neurospheres may not reflect a natural pool of neural precursors because their growth conditions select for responders to non-physiologically high concentrations of growth factors and unattached growth [27, 28]. It is thus possible that by the beginning of co-culturing, cells derived from neurospheres may have been reprogrammed by the culture conditions. Furthermore, in neurosphere co-culture experiments the state of growth of neural stem cell progenitors is difficult to observe because it occurs in a “blind spot” within the neurospheres themselves. Finally, induction of neural differentiation through the change of cell attachment status may obscure the effects of test substances, in the neurosphere system.
For the reasons stated above, systems capable of quantifying the effects of neurogenic and gliogenic factors on neural precursor cells, neurons, astrocytes and oligodendrocytes under the same conditions have not been available.
SB623 cells are derived from MSC by transfecting MSC with a vector encoding a Notch1 intracellular domain. See, e.g., U.S. Pat. No. 7,682,825. Previous work has shown that ECM produced by human MSC, and SB623 cells derived therefrom, effectively supports the growth and differentiation of rat embryonic cortical cells without added factors or serum (29, see also US 2010/0310529, the disclosure of which is incorporated by reference in its entirety for the purpose of describing certain properties of the ECM produced by MSC and SB623 cells).
As set forth above, there remains a need for a simple and accurate in vitro system that models the interactions of substances possessing neurogenic and/or gliogenic activity (e.g., MSC and their derivatives, e.g., SB623 cells) with complex populations of neural cells, and quantifies the potency of such substances.
Provided herein are in vitro systems for co-culture of MSC, and/or their derivatives (e.g., SB623 cells), with neural cell populations under conditions that optimize the ability to quantitate the effects of factors that influence the growth and differentiation of the different neural cells in the culture.
These culture systems can be used to provide quantitative functional assays for measuring the effects of substances (e.g., MSC and their derivatives, e.g., SB623 cells, conditioned medium, polypeptides, organic compounds) on various types of neural cells (e.g. neurons, astrocytes, oligodendrocytes). In particular, neurotrophic, neurogenic, gliotrophic and gliogenic factors, and sources of such factors, can be identified and quantitated.
Using the assays described herein, a number of substances having neurogenic and gliogenic activity have been identified.
Accordingly, the present disclosure provides, inter alia, the following embodiments.
(a) culturing mesenchymal stem cells (MSC) on a solid substrate;
(b) removing the MSC from the substrate, such that an extracellular matrix produced by the MSC remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of neurons;
(a) culturing mesenchymal stem cells (MSC) on a solid substrate;
(b) removing the MSC from the substrate, such that an extracellular matrix produced by the MSC remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of glial cells;
(a) culturing cells on a solid substrate, wherein the cells are descendants of mesenchymal stem cells that have been transfected with a nucleic acid encoding a Notch intracellular domain;
(b) removing the cells from the substrate,
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of neurons;
(a) culturing cells on a solid substrate, wherein the cells are descendants of mesenchymal stem cells that have been transfected with a nucleic acid encoding a Notch intracellular domain;
(b) removing the cells from the substrate, such that an extracellular matrix produced by the cells remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of glial cells;
(a) culturing mesenchymal stem cells (MSC) on a solid substrate;
(b) removing the MSC from the substrate, such that an extracellular matrix produced by the MSC remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of neural precursor cells;
(a) culturing cells on a solid substrate, wherein the cells are descendants of mesenchymal stem cells (MSC) that have been transfected with a nucleic acid encoding a Notch intracellular domain;
(b) removing the cells from the substrate, such that an extracellular matrix produced by the cells remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring growth of NPC;
(a) culturing mesenchymal stem cells (MSC) on a solid substrate;
(b) removing the MSC from the substrate, such that an extracellular matrix produced by the MSC remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring differentiation of NPC;
(a) culturing cells on a solid substrate, wherein the cells are descendants of mesenchymal stem cells (MSC) that have been transfected with a nucleic acid encoding a Notch intracellular domain;
(b) removing the cells from the substrate, such that an extracellular matrix produced by the cells remains on the substrate;
(c) culturing embryonic cortical cells on the substrate of step (b);
(d) adding a substance to the culture of step (c); and
(e) measuring differentiation of NPC;
It has proven difficult to establish in vitro culture conditions that will support the growth and differentiation of the various different types of neural cells. The present inventors have devised an in vitro culture system in which neural precursor cells, neurons, astrocytes and oligodendrocytes are all able to grow and differentiate. The culture system disclosed herein thus allows, for the first time, quantitative evaluation of the effect of a test substance on the growth and differentiation of neural cells. The system comprises a culture of neural cells (e.g. embryonic rodent cortical cells) on an extracellular matrix in the presence of a test substance, followed by analysis of the neural cell culture for the expression of one or more marker molecules. The extracellular matrix used in these assays is produced by (a) a mesenchymal stem cell, or (b) a mesenchymal stem cell that has been transfected with a nucleic acid, wherein the nucleic acid encodes a Notch intracellular domain but does not encode full-length Notch protein (e.g., a SB623 cell).
Various aspects of this system contribute to its ability to provide quantitative information on the potency of various neurogenic and gliogenic factors. In one aspect, the neural cells are cultured on an extracellular matrix produced by MSC or SB623 cells (cells that have been derived from MSC by transfecting MSC with a vector containing sequences encoding an Notch intracellular domain). In another aspect, the amount of time that the neural cells are co-cultured with a test substance is chosen to optimize detection and quantitation of the marker that is being assayed. The duration of co-culture prior to assay is unique to each marker. For example, co-culture is conducted for five days for measurement of nestin and CNPase; and for seven days for measurement of GFAP, DCX and MAP2. In yet another aspect, the concentration of cells in the culture is optimized. For example, neural cells are used at a concentration of 1.5×104 cells/ml; MSC and SB623 cells are used at a concentration of 0.5-1.5×103 cells/ml.
The quantitative assay system disclosed herein utilizes ECM from mesenchymal cells such as MSC and their derivatives (e.g., SB623 cells) as a biological substrate for co-cultures of test substances (e.g., MSC or their derivative SB623 cells, conditioned medium, growth factors, cytokines) and neural cell populations, and provides a culture system that is favorable to the growth of both mesenchymal cells and neural cells. Such a system, in turn, allows quantitation of the effects of mesenchymal cells, as well as effects of other cells and substances, on the growth and differentiation of various types of neural cells.
The advantages of the assays described herein include that fact that developmental transitions occur under physiological conditions and over a physiological time-course, rather than in response to abnormal physical conditions, such as attachment or aggregation (cf. neurosphere cultures). In addition, the stage of development of the cells being assayed can be easily determined, as development does not occur in the interior of a neurosphere. Finally, the assays disclosed herein do not require external growth factors; thus allowing the effects of such factors to be quantitated in this system.
Using this system, the inventors have determined that not only does mesenchymal cell ECM support the growth of neural cell populations (such as, for example, embryonic cortical cells), but that addition of MSC or SB623 cells to neural cell populations growing on mesenchymal cell ECM substantially enhances growth and differentiation of all neural lineages (e.g., neurons, astrocytes and oligodendrocytes).
Compared to existing co-culture systems, much lower ratios of mesenchymal cells to neural cells are capable of inducing significant growth and differentiation of neural cells in the ECM-based co-cultures described herein. For example, the assay systems described herein are sensitive enough to detect the effect of approximately 50 mesenchymal cell on 5,000 neural cells.
Provided herein are quantitative assays for neurogenic and gliogenic factors, as well as factors that promote the growth and differentiation of neural precursor cells. The assays can also be used to identify and quantitate sources of such factors, such as cell cultures or conditioned media.
To conduct the assays, MSC or SB623 cells (referred to collectively as “mesenchymal cells”) are grown in a vessel, such as a tissue culture dish, for a period of time sufficient for the cells to lay down an extracellular matrix on the surface of the vessel. Any solid substrate can be used as a surface on which the cells are grown, as long as it supports the growth of the cells and the elaboration of an extracellular matrix by the cells. Suitable substrates include plastic, glass or nitrocellulose. Further, the substrate may be coated with a substance such as, for example, fibronectin or collagen, or a reconstituted basement membrane such as, for example, Matrigel™.
An example of a suitable substrate is a plastic tissue culture dish or flask. The cells can be grown for one day, two days, three days, one week, two weeks, one month, or any time interval therebetween as desired. For additional details on ECM elaborated by MSC and SB623 cells, see U.S. Patent Application Publication No. 2010/0310529, the disclosure of which is incorporated by reference for the purpose of describing ECM elaborated by MSC and SB623 cells (denoted “differentiation-restricted descendants of MASCs” in that publication) and its properties.
MSC can be obtained by selecting adherent cells from bone marrow samples. Bone marrow can be obtained commercially (e.g., from Lonza, Walkersville, Md.) or from bone marrow biopsies. Other sources of mesenchymal stem cells include, for example, adipose tissue, dental pulp, cord blood, placenta and the decidua. MSC can be obtained from any animal, including mammals, and including humans.
Exemplary disclosures of MSC are provided in U.S. patent application publication No. 2003/0003090; Prockop (1997) Science 276:71-74 and Jiang (2002) Nature 418:41-49. Methods for the isolation and purification of MSC can be found, for example, in U.S. Pat. No. 5,486,359; Pittenger et al. (1999) Science 284:143-147 and Dezawa et al. (2001) Eur. J. Neurosci. 14:1771-1776. Human MSC are commercially available (e.g., BioWhittaker, Walkersville, Md.) or can be obtained from donors by, e.g., bone marrow aspiration, followed by selection for adherent bone marrow cells. See, e.g., WO 2005/100552.
SB623 cells are derived from MSC by transfecting MSC with a vector containing sequences that encode a Notch intracellular domain (NICD) but do not encode the full-length Notch protein, such that the transfected cells express exogenous NICD but do not express exogenous full-length Notch protein. Methods for obtaining MSC, and for deriving SB623 cells from MSC populations, are described, for example, in U.S. Pat. No. 7,682,825 and in US Patent Application Publication No. 2010/0266554, the disclosures of which are incorporated by reference for the purposes of describing MSC and SB623 cells, and methods of obtaining these cells.
Subsequent to growth on the substrate for a predetermined amount of time, the MSC or SB623 cells are removed from the substrate, leaving behind an extracellular matrix deposited on the substrate. Methods of removing cells from a substrate are well known in the art. In the practice of the methods disclosed herein, removal of cells from the substrate must be sufficiently gentle that the ECM that has been elaborated by the cells remains on the substrate. Such methods include, for example, treatment with non-ionic detergent (e.g., Triton X-100, NP40) and alkali (e.g. NH4OH). See the “Examples” section infra for additional details.
The ECM-containing substrate is then used as a substrate for co-culture of neural cells and one or more test substance(s), and the effect of the test substance(s) on the neural cells is determined and quantitated. Introduction of the neural cells and the test substance to the culture can be simultaneous, or in either order.
Any type of neural cell or neural cell population can be used; such cells are known in the art. A convenient source of neural cell populations are rodent embryonic cortical cells (e.g., from rat or mouse), which can be obtained commercially (BrainBits, Springfield, Ill.). In certain embodiments, the neural cell population is enriched in neural precursor cells.
A test substance can be any chemical compound, macromolecule (e.g., nucleic acid or polypeptide), cell, cell culture, cell fraction or tissue, or combination thereof. For example, growth factors and cytokines, low molecular weight organic compounds, mRNA molecules, siRNA molecules, shRNA molecules, antisense RNA molecules, ribozymes, DNA molecules, DNA or RNA analogues, proteins (e.g., transcriptional regulatory proteins), antibodies (e.g., neutralizing antibodies), enzymes (e.g., nucleases), glycoproteins, glycans, proteoglycans, cells, cell membrane preparations, cell cultures, conditioned medium from cell cultures, subcellular fractions and tissue slices or tissue fractions are all suitable test substances. Test substances can also include electromagnetic radiation such as, for example, X-rays, light (e.g., ultraviolet, infrared) or sound (e.g., subsonic or ultrasonic radiation). In certain embodiments, the combination of a protein and a neutralizing antibody to the protein is used as a test substance.
Naturally-occurring test substances can include soluble molecules (e.g., proteins) synthesized and secreted by cells, as well as molecules (e.g., proteins) that are synthesized by a cell, transported to the cell surface, and remain embedded in the cell surface, with all or a portion of the molecule exposed to the exterior of the cell (i.e., surface molecules, surface proteins or surface glycoproteins).
Neural cells and test substances are co-cultured for an appropriate amount of time, as determined by the practitioner of the method. For example, co-culture can be conducted for 1 hour, two hours, three hours, four hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, one month, or any time interval therebetween.
The effect(s) of the test substance(s) on the neural cells is determined by measuring the expression of one or more markers in the neural cells. Depending on the marker or markers chosen, it is possible to assay for formation of neural precursor cells, neurons, astrocytes, or oligodendrocytes.
In one embodiment, the effect of a particular protein, either native or recombinant, on neurogenesis or gliogenesis can be determined by adding the protein to a culture of neural cells growing on a MSC or SB623 ECM and assaying for the appropriate neuronal or glial marker. Optionally, a low concentration (1%, 2%, 5%, 10%, 20%, 30%, 40%, 50% or any value therebetween) of conditioned medium from MSC or SB623 cells can also be included in the culture. For example, inclusion of conditioned medium can provide additional factors required for the process under study, other than the one being tested, thereby allowing the effect of one component of a multi-factor signaling system to be assessed.
Molecular and morphogenetic markers for neural precursor cells, neurons, astrocytes and oligodendrocytes are well-known in the art; the following are provided as examples.
Markers for neural precursor cells include, for example, nestin, glutamate transporter (GLAST), 3-phosphoglycerate dehydrogenase (3-PGDH, astrocyte precursors), ephrin B2 (EfnB2), Sox2, Pax6, and musashi. In certain embodiments, proliferative capacity can also be used as a marker for neural precursor cells. Proliferative capacity can be measured, for example, by incorporation of bromodeoxyuridine, carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, expression of Ki-67 or expression of proliferating cell nuclear antigen (PCNA).
Markers for neurons include, for example, microtubule-associated protein 2 (MAP2), β-tubulin isotype III (also known as β-III tubulin and TuJ-1), doublecortin (DCX), neurofilament proteins (e.g., neurofilament-M), synaptophysin, and neuron-specific enolase (also known as enolase-2 and gamma enolase). Neurite outgrowth can also be used as a marker for neuronal development.
Additional neuronal markers are listed in the following table:
Glial fibrillary acidic protein (GFAP), glutamate transporter (GLAST), 3-PGDH and glutamine synthetase can be used as markers for astrocytes.
Markers for oligodendrocytes include, for example, the A2B5 antigen, galactocerebroside (GalC), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase), the O1 antigen, the O4 antigen, myelin basic protein, oligodendrocyte transcription factor 1, oligodendrocyte transcription factor 2, oligodendrocyte transcription factor 3, NG2, and myelin-associated glycoprotein.
Expression of markers can be measured by techniques that are well-known in the art. For example, expression of proteins can be measured and quantitated by immunofluorescence, immunohistochemistry (IHC), ELISA and protein blotting (e.g., Western blots).
Expression of mRNA can be measured and quantitated by methods including, for example, blotting, nuclease protection and reverse transcription-polymerase chain reaction (RT-PCR).
Depending on the test substance and marker being assayed, it may be necessary to ensure that the assay is specific for the molecule produced by the neural cell and does not cross-react with the same or a similar molecule produced by the test substance, especially if the test substance is a cell, such as a MSC or a SB623 cell. For example, MSC express nestin; therefore, if nestin is being assayed as a marker for a neural precursor cell in a co-culture of rat cortical cells and human MSC, an antibody specific for rat nestin is used in the assay. Similarly, if nucleic acid expression is assayed, such as by quantitative reverse transcription/polymerase chain reaction (qRT-PCR) or TaqMan, species-specific primers (and probes, if applicable) are used.
In certain embodiments, the expression of a marker by a neural cell in the assay described above (i.e. co-culture of neural cells and test substance) is compared to the expression of the same marker in neural cells in the absence of the test substance(s).
The assays described herein can also be used to quantitate the differentiation of neural precursor cells (NPCs) by measuring expression of markers characteristic of the progeny of NPCs, which include neurons, astrocytes and oligodendrocytes. Such markers are well-known in the art and exemplary markers have been described herein.
In certain embodiments, a kit for assaying the neurogenic or gliogenic potential of a test substance, or for assaying the ability of a substance to promote the growth and/or differentiation of neuronal precursor cells, is provided. The kit contains one or both of MSC and SB623 cells (optionally in a cryopreserved state), along with one or more culture vessels, and optionally culture medium, to allow the user to grow the MSC or SB623 cells on the culture vessel. The kit may also contain reagents (e.g., nonionic detergents such as Triton X-100 or Nonidet P-40; ammonium hydroxide) for removing the MSC or SB623 cells from the culture vessel so as to leave an extracellular matrix deposited on the surface of the culture vessel. The kit can also contain a sample of neural cells (e.g., rat E18 cortical cells). Labeled antibodies to various neuronal and glial markers may also be included in the kit; and oligonucleotide probes and/or primers specific for mRNAs encoding neuronal and glial markers can also be included. Any type of reagent that will detect a neuronal or glial marker (e.g., a protein) or its encoding mRNA can be included in the kit. Reagents and/or buffers and/or apparatus suitable for immunohistochemistry, FACS, RT-PCR, electrophysiology and pharmacology can also be included in the kit.
In additional embodiments, a kit as disclosed herein can contain one or more culture vessels with an ECM from MSC or SB623 cells deposited thereon. Such a kit can optionally include neural cells and reagents (e.g., antibodies, probes, primers) to detect neuronal and/or glial markers. Such a kit can also optionally include reagents and/or buffers suitable for immunohistochemistry, FACS, RT-PCR, electrophysiology and pharmacology.
In additional embodiments, a kit can contain purified extracellular matrix from MSC or SB623 cells (or a mixture thereof), for application to a culture vessel.
In further embodiments, a kit comprises a solid substrate (e.g., a culture vessel) with a biological layer deposited thereon, wherein the biological layer is an extracellular matrix deposited by:
(a) a mesenchymal stem cell, or
(b) a mesenchymal stem cell that has been transfected with a nucleic acid, wherein the nucleic acid encodes a Notch intracellular domain but does not encode full-length Notch protein.
In the operation of the kits, neural cells are grown in contact with an extracellular matrix from MSC or SB623 cells, in the presence of a test substance, and the neural cells are analyzed for the expression of a chosen neuronal or glial marker. With certain of the aforementioned kits, deposition of the ECM on a culture vessel (by MSC and/or SB623 cells) and removal of the cells that elaborated the ECM, is conducted by the user prior to adding the neural cells and the test substance to the culture vessel.
MSC and SB623 cell preparation has been described [29]. Briefly, human adult bone marrow aspirates (Lonza, Walkersville, Md.) were grown in αMEM (Mediatech, Herndon, Va.) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah), 2 mM L-glutamine, and penicillin/streptomycin (both from Invitrogen, Carlsbad, Calif.). On the second passage, some cells were cryopreserved (MSC preparation) and some cells were plated for the preparation of SB623 cells. For SB623 cell preparation, MSC were transfected with a pCI-neo expression plasmid encoding the human Notch1 intracellular domain (NICD). After one day of culture, transfected cells were placed under selection with G418 (Invitrogen) for 7 days, after which selection was removed and the cultures were grown and expanded by passaging twice. SB623 cells were then harvested and cryopreserved using Cryostor CS5 (BioLife Solutions, Bothell, Wash.). Cells from 3 different donors were used in the studies described herein. MSC and SB623 cells were thawed and washed once with αMEM before use. For co-culture experiments, cells were then resuspended in a neural growth medium consisting of Neurobasal medium supplemented with 2% B27 and 0.5 mM GlutaMAX (all from Invitrogen). For the production of ECM coating or the production of conditioned medium (CM), cells were plated in αMEM supplemented with 10% FBS and penicillin/streptomycin.
For the preparation of wells coated with ECM, SB623 cells were plated at 3×104 cells/cm2 in 96-well plates or on glass cover slips (Fisher Scientific, Pittsburgh, Pa.) which were placed into 12-well plates (all plates were purchased from Corning Inc, Corning, N.Y.) and grown for 5 days. Subsequently the medium was changed to serum-free, and the cells were cultured for an additional 2 days. Cells were then removed from the ECM using a protocol described previously [29] with some modifications. Briefly, cells were treated with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, Mo.) in water at room temperature for 40 min; then cell lysates were carefully aspirated, and a 1:100 (v/v) solution of concentrated NH4OH (Sigma-Aldrich) in water was slowly added for 5-7 min, then removed. For washing, the wells were filled completely with PBS and incubated for at least 3 hours. Wells were either used immediately or stored at 4° C.
MSC or SB623 cells were plated at 3×104 cells/cm2 and grown in αMEM supplemented with 10% FBS and penicillin/streptomycin for 3-4 days until confluence. Then the medium was replaced with Neurobasal medium (Invitrogen), and the cultures were incubated for 1-2 hours. This medium was discarded and replaced with fresh Neurobasal medium, using half of the volume typically used for cell growth. The cells were incubated for 24 hours, after which the medium was collected, and particulate matter was removed by centrifugation. The medium was dispensed in aliquots and stored at −70° C. MSC-CM preparations were supplemented with 2% B27 and 0.5 mM GlutaMAX before use.
Rat embryonic (E18) brain cortex pairs were purchased from BrainBits (Springfield, Ill.); and a cell suspension was prepared as described [29]. Briefly, cortices were incubated with 0.25% Trypsin/EDTA at 37° C. for 5-7 min, and trypsin was removed. The tissue was washed with αMEM containing 10% FBS, then with PBS. DNase (MP Biomedicals, Solon, Ohio) at 0.25 mg/ml was then added, and the contents of the tube were mixed by vortexing for 30 sec. The resulting cell suspension was triturated, diluted with PBS, pelleted and then resuspended in neural growth medium (described above).
Plates, coated as described above, were pre-warmed with a portion of neural growth medium, and then varying numbers of mesenchymal cells were added. Subsequently, neural cells were added at a density of 1.5×104 cells/cm2 to all but control wells, and cultures were incubated for the indicated time periods. For each time point, a separate plate was used that included quadruplicate samples. For quantitation, cells were plated in 96-well plates and MSC or SB623 cells were added at decreasing densities, starting at 1.5×103 cells/cm2 (i.e., 500 cells per well) For immunostaining, cultures were plated on ECM-coated cover slips in 12-well plates. MSC or SB623 cells were added at a constant cell density of 1.5×103 cells/cm2 unless indicated otherwise.
In a subset of experiments, in which the effects of cells (MSC or SB623) were compared to the effects of their conditioned medium, a cryopreserved aliquot of cells was used to generate the conditioned medium prior to an experiment, and an aliquot of cells from the same donor was thawed on the day of experiment to generate a corresponding cell suspension. Cells were applied at decreasing concentrations as described above and conditioned medium was used at decreasing concentrations starting from 50% of total medium. For quantitation of gene expression, all culture conditions were tested on the same PCR plate using the same standard curve for each neural marker.
Medium was not changed during co-culture experiments (which lasted for 7-8 days in 96-well format and for up to 14 days with the cells on cover slips). No signs of culture decline were noticed and the viability of neural cells was above 95% when assessed on the last day of culturing using Trypan Blue exclusion.
In another set of experiments, when cells were co-cultured under non-adherent conditions, Ultra-Low Adhesion Costar 12- or 24-well plates were used. Mesenchymal and neural cells were mixed in the indicated quantities and plated in neural growth medium. As a control, neural cells were grown alone or in the presence of 20 ng/ml or 50 ng/ml each of EGF and FGF2 purchased from either R&D Systems (Minneapolis, Minn.) or Peprotech (Rocky Hill, N.J.). Medium was not changed over the course of the experiment (2 weeks).
Cultures that were grown on glass cover slips were fixed with 4% paraformaldehyde (Electron Microscopy Science, Hatfield, Pa.) for 20 min, washed once with PBS and incubated for 30 min in blocking solution containing 10% normal donkey serum (Jackson Immunoresearch, West Grove, Pa.), 1% bovine serum albumin (Sigma-Aldrich), 0.1% Triton X-100. Then a goat polyclonal antibody against rat Nestin (R&D Systems, Cat #AF2736) was added into the blocking solution at 1:1000 and incubated overnight at 4° C. Cover slips were washed with PBS and then either rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (Dako, Denmark) (1:2000), mouse monoclonal anti-microtubule-associated protein 2 (MAP2) (Sigma-Aldrich) (1:1000), or mouse monoclonal anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) (Millipore, Billerica, Mass.) (1:200) was added and the cover slips were incubated for 1 hour at room temperature. After washing, cover slips were incubated for 1 hour with secondary antibodies: DyLight 488-conjugated AffiniPure donkey anti-goat F(ab′)2 fragments of IgG (1:1000) in combination with either DyLight 549 488-conjugated AffiniPure anti-rabbit F(ab′)2 fragments of IgG (1:2000) or Cy3-conjugated AffiniPure donkey anti-mouse IgG (1:1000), all from Jackson Immunoresearch, and all selected for use in multiple labeling by the manufacturer. After washing with PBS and water, the slips were mounted with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
In some experiments, prior to fixation, cells were incubated for 7-8 hours with 10 uM 5-bromo-2′-deoxyuridine (BRDU, from Sigma-Aldrich) with or without mitomycin at a concentration of 50 ug/ml. Cultures then were fixed with 2% PFA, permeabilized with 0.5% Triton and treated with deoxyribonuclease (MP Biomedicals, Solon, Ohio) in a buffer containing 0.15 M NaCl and 4.2 mM MgCl2 for 1 h at 37° C. The cultures were then post-fixed with cold methanol (Fisher Scientific, Fair Lawn, N.J.) for 10 min. After blocking as described above, the cultures were incubated with anti-BRDU monoclonal antibody (BD Pharmingen), then with the anti-mouse secondary antibody described above, then with Alexa Fluor-conjugated TUJ1, a Neuronal Class III β-Tubulin-specific antibody (Covance, Princeton, N.J.).
Fluorescence microscopy was conducted using a Nikon Eclipse50i (Nikon Instruments, Melville, N.Y.) and a Nikon Digital Camera DXM1200C.
Under the conditions described herein, none of antibodies reacted with the mesenchymal cells.
Growth and culture of cells, for quantitation of expression of mRNAs encoding various neural markers, was conducted in 96-well plates. After culturing for the indicated time, the culture medium was carefully and completely aspirated using a Nunc ImmunoWasher equipped with 10 ul pipette tips; and cells were lysed with 20 ul/well of lysis buffer, either Cell-to-Signal™ (Applied Biosystems/Ambion, Austin, Tex.) or SideStep™ (Agilent Technologies, Santa Clara, Calif.) for 3 min. Then the lysates were carefully pipetted up and down and samples (in quadruplicate) were combined pair-wise (thus making biological duplicates), transferred to a storage plate and frozen at −70° C.
For gene expression testing, samples were thawed and aliquots were diluted 1:10 with PCR-grade water. A sample with a high expected expression level was also used to prepare serial dilutions in 10% lysis buffer to serve as a series of standards for the quantification. The diluted samples were used as templates in one-step qRT-PCR reactions, in combination with QuantiTect® Probe RT-PCR Master Mix from Qiagen (Valencia, Calif.) and TaqMan® gene expression assays purchased from Applied Biosystems (Foster City, Calif.). The absence of cross-reaction between corresponding mRNAs from rat and human cells was established in experiments involving rat neural cells, as well as human mesenchymal and neural cells. The following pre-optimized assays, all designed across exon-exon boundaries, were used: rat-specific—nestin (Rn00564394_m1), MAP2 (Rn00565046_m1), GFAP (Rn00566603_m1), CNPase (Rn01399463_m1), Doublecortin (Dcx)(Rn00584505_m1), glyceraldehyde 3-phosphate dehydrogenase (rGAP)(Rn-1462661_g1); and human-specific—glyceraldehyde 3-phosphate dehydrogenase (huGAP) (4333764F), bone morphogenetic protein 4 (BMP4) Hs00370078_m1, and fibroblast growth factor 2 Hs00266645_m1. Numbers in parentheses refer to the manufacturer's (Applied Biosystems) assay ID numbers. For amplification reactions, a LightCycler 480 (Roche, Mannheim, Germany) was programmed according to the Master Mix manufacturer's protocol, with 40-60 amplification cycles. Analysis was done using a Second Derivative Maximum method.
Neural cells were plated at 1.5×104 cells/cm2 alone or together with either MSC or SB623 cells at 1.5×103 cells/cm2 on ECM-covered glass cover slips in neural growth medium containing 10-fold less B27 than normally used (0.2%), and allowed to grow for 18-24 hrs. Then the cultures were fixed and stained for MAP2 expression and mounted with DAPI-containing medium, as described above. Approximately 12-15 fields were photographed using the same exposure time, and a total of 20 neurons per condition were analyzed. The length of the longest neurite was measured for each cell and the number of neurites per cell was counted.
The composition of ECM-based neural cultures grown with or without mesenchymal cells was first analyzed using immunocytochemistry for Nestin, a marker of neural stem cells/early progenitors, and MAP2, a neuronal marker. At various times cultures were fixed and incubated with rat-specific anti-Nestin antibody and anti-MAP2 antibody. All cultures were also counterstained with the nucleus-specific dye DAPI. Fixed and stained cultures were then examined by fluorescence microscopy. On day 1, single positive Nest+ and MAP2+ cells were present in all cultures tested, along with some MAP2+Nes+ double-positive cells, in which MAP2 staining and Nestin staining were co-localized. There was also a small fraction of double-negative cells. By day 3, no double-positive cells were detected, while single-positive cells extended their processes. On day 5, MAP2+ neurons continued to extend neurites, while Nes+ cells were significantly increased in numbers. At this time point, Nes+ cells formed colonies. A greater number of colonies, and larger colony size, were observed in the presence of mesenchymal cells. Double-positive cells were not detected.
By day 9, a large number of MAP2+Nes+ double-positive cells were observed, as well as MAP2+Nes− neurons and MAP2−Nes+ cells. The double-positive cells were found in greater numbers in co-cultures with SB623, compared to co-cultures with MSC, while cultures without mesenchymal cells had the smallest number of MAP2+Nes+ cells. These double-positive cells had nuclei of a characteristic bilobular shape, thin MAP2-positive processes, and a strong Nestin reactivity localized to a perinuclear area—most frequently, to a cleft between two nuclear lobes situated adjacent to the most prominent outgrowth. This morphology resembled that of the MAP2+Nes+ double-positive cells present on the first day of culturing.
When neural cells and mesenchymal cells were co-cultured on PDL, approximately the same frequency of double-positive and single-positive cells were observed on Day 1 of culture as were observed when cells were co-cultured on ECM. At later time points, no developed single-positive Nes+ cells were detected (very rare Nes+ cells were round, with dense nuclei). By Day 5 of culture, only a small number of double-positive cell colonies, each consisting of very few cells, were observed. These results indicate that, on PDL, most or all Nes+ cells were committed to neuronal differentiation. At day 9, double-positive colonies were more prominent in the presence of mesenchymal cells than in their absence.
The status of mesenchymal cells in co-cultures was also examined using phase contrast microscopy and staining for α-smooth muscle actin, a mesenchymal marker. On PDL, mesenchymal cells were barely spread and usually disappeared around day 5-7. On ECM, mesenchymal cells were easily detected throughout the duration of co-culturing; they appeared well-spread, moving, and appeared to be slowly proliferating.
Neuritogenesis was very active on both ECM and PDL in co-cultures, but was further enhanced in the presence of mesenchymal cells during first 18-24 hrs in culture. To increase this differential response, cultures were plated in neural growth medium with a low concentration of B27 supplement. Under these conditions, longer neurites were observed in the presence of MSC or SB623 cells after 24 hours of co-culturing, than in cultures of neural cells alone (Table 1). However, no significant difference in numbers of neurites was noticed (Table 1).
Astrocyte development was assayed by immunohistochemical analysis for expression of glial fibrillary acidic protein (GFAP). Double-staining of ECM-based cultures for glial fibrillary acidic protein (GFAP, an astrocyte marker) and Nestin revealed the absence of GFAP reactivity before day 3 in all cultures. Around day 5, GFAP-expressing cells began to be observed in co-cultures within Nes+ colonies as single- or double-positive cells. GFAP-expressing cells were not observed in cultures not containing mesenchymal cells. Different colonies had variable proportions of GFAP+Nes+ cells and the more fully differentiated GFAP+Nes− cells. No GFAP+ cells were detected at this time in cultures lacking mesenchymal cells. On day 9, all three phenotypes (GFAP+Nes+, GFAP−Nes+, and GFAP+Nes−) were present in all cultures, with GFAP+Nes− cells predominating.
With respect to its intracellular localization, GFAP immunoreactivity appeared initially (at day 5) as intercalating filaments beside or within Nestin filaments in some Nestin-positive filamentous cells. Later, Nestin was practically displaced by GFAP in certain cells, as evidenced by a patchy distribution of Nestin staining, while other cells continued to express Nestin only. In co-cultures, GFAP+Nes+ cells had generally the same morphology as GFAP+Nes− cells, while among GFAP−Nes+ cells, morphology varied. One type of cell had very long processes, frequently exceeding 200 μm. Other types were small GFAP−Nes+ cells, with Nestin reactivity localized eccentrically with respect to the nucleus, either as a “lace” from one side of nucleus, or concentrated in a cleft of a bilobular nucleus and extended into a process positioned against the cleft. The latter morphology was similar to that of Nes+MAP2+ cells described in the previous example.
No GFAP+ cells were detected when co-cultures were conducted on PDL.
Oligodendrogenesis was assessed by immunohistochemical analysis of expression of the early oligodendrocyte marker 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase). CNPase+ cells could not be detected before day 9 of co-culture. On day 12, in cultures grown on ECM in the absence of mesenchymal cells, CNPase reactivity could be detected only in a very few, usually dividing, cells. At the same time point, in co-cultures with mesenchymal cells, CNPase+ cells appeared in clusters, with CNPase expression localized to the perinuclear area. In co-cultures with SB623 cells, CNPase staining was both more intense and more extensive, extending throughout the cytoplasm. Expression of CNPase did not co-localize with Nestin expression.
No CNPase+ cells were detected when co-cultures were conducted on PDL.
Proliferation of neural cells was assayed in cultures containing 1.5×104 neural cells/cm2, with or without MSC at 1.5×103 cells/cm2, using two methods. In the first method, DAPI-stained nuclei were counted on slides prepared for immunochemistry analysis as described above. Five microscopic fields at 200×-magnification were counted and averaged per condition, from 2 experiments. Extremely condensed or fragmented nuclei were considered to be indicative of dead cells. MSC nuclei were excluded from counting based on their distinctive size.
In the second method, neural cell proliferation was measured in microplate format co-cultures using a quantitative PCR assay for rat noggin, a single-exon gene (Rn01467399_s) (Applied Biosystems). The analysis was conducted as described for qRT-PCR, with the exception that the reverse transcription step of the qRT-PCR protocol was omitted. Minimal amplification of human noggin sequences from the MSC was detected (
The results are shown in
Proliferation of neural precursor cells was also assayed in co-cultures of rat neural cells with MSC on ECM. On day 7 of co-culture, cultures were treated with BRDU for 7 hours following by fixing and immunostaining with antibody to BRDU and with the neuron-specific TUJ1 antibody. Irrespective of whether neural cells were cultured alone or co-cultured with MSC, at this time point the cultures contained small cells, with barely developed processes, exhibiting reactivity with both anti-TUJ1 and anti-BRDU antibodies, indicative of a proliferating neural precursor cell. Quantitation of these neural precursor cells, using a PCR assay for the rat noggin gene, showed that their numbers were increased when the neural cells were co-cultured with MSC (
In this example, the time course of expression of various neuronal (doublecortin, MAP2), neural precursor (nestin) and glial markers (GFAP and CNPase), in neural cells cultured on ECM, was analyzed in the presence and absence of 200 MSC/well. Samples were collected at various time points and frozen. All samples were subsequently thawed and assayed in parallel by qRT-PCR. Primers, probes and amplification conditions were as described above. The results are shown in
Based on these results, the optimal time for detection of Nestin and CNPase mRNA expression was determined to be Day 5 of co-culture; and the optimal time for detection of DCX, MAP2 and GFAP mRNA expression was determined to be Day 7 of co-culture.
For quantitative assays, rat cortex cells (5000 cells/well) were cultured alone or co-cultured with decreasing numbers of MSC, from 500 to 32 cells/well. As a control, MSC were also cultured alone at 500 cells/well. qRT-PCR Taqman assays for rat Nestin, MAP2, GFAP, and CNPase mRNAs were used to quantify gene expression. A human-specific glyceraldehyde 3-phosphate dehydrogenase (huGAP) qRT-PCR Taqman assay was used to estimate MSC numbers.
To observe a MSC-dependent dose response using Nestin, MAP2, or CNPase as markers, the timing of sampling was important. For example: the optimal timing for testing rat Nestin gene expression was between day 4 and 6, since on day 7 the expression reached saturation.
Rat CNPase mRNA expression was first detected around day 5, long before the protein could be detected. At Day 5, CNPase mRNA levels were directly proportional to MSC concentration in co-cultures. After day 5, CNPase gene expression levels continued to increase; but by Day 7, the MSC-dose-dependence curve became biphasic (
Expression of GFAP mRNA showed a strong and consistent dose response to mesenchymal cell concentration, as soon as it could be detected (day 4 or 5) and did not demonstrate saturation for the remainder of the culture period (days 7-9).
In this example, the effects of live MSC on neural cell differentiation were compared to the effects of their conditioned medium (CM); and the effects of using ECM as a substrate were compared to the use of poly-D-lysine.
Quantitative neural differentiation assays (qRT-PCR) were used to determine which components of mesenchymal stem cell cultures had the greatest neuropoietic effects. For this purpose, the response of neural cells to MSC was compared to that to MSC-CM, and cells were co-cultured either on ECM- or PDL-coated plates. Decreasing concentrations of MSC and MSC-CM were used to ensure that effects were analyzed below saturation. The results are summarized in Table 2. To simplify the presentation, the table includes only the data from experiments that included the highest concentration of mesenchymal cells (500 cells/well) or MSC-CM (50%). Lower concentrations of these additives stimulated lower marker expression levels, confirming that the responses were neither saturated nor down-regulated. The results were expressed relative to the levels in cultures grown on ECM without additives on the indicated day.
Similar levels of rMAP2 expression on day 1 were observed under all test conditions indicating that initial neuron attachment and development was similar. Later, on day 5, the presence of either live MSC or MSC-CM increased the expression of this marker 2-3-fold on either substrate. Nestin gene expression was 2-3 orders of magnitude stronger on ECM than on PDL. On day 5, Nestin gene expression was substantially increased in the presence of both MSC-CM and live MSC on both ECM and PDL. CNPase gene expression on day 5 was induced by live MSC and induced even more strongly by MSC-CM on ECM-based cultures, while on PDL-based cultures, it was below detection limits. CNPase gene induction could be detected on PDL on day 7, with MSC-CM being a more effective stimulus than live MSC. GFAP gene expression was induced on ECM most dramatically by live MSC and, to a lesser extent, by MSC-CM. On PDL, GFAP expression was below quantification limits throughout the study.
Human GAP expression was also tested on day 1 and on day 7 of this study. On day 1, human GAP gene expression in co-cultures conducted on PDL-coated plates was only slightly lower than in cells cultured on ECM, while on day 7 it was below quantification limits. Microscopic examination revealed that, after 7 days on PDL, only a few mesenchymal cells survived, and they were barely spread, while on ECM they exhibited their typical morphology and had slightly increased in number. The results are summarized in Table 3.
1 (6%)
1 (1%)
# Relative to the “No add, ECM” sample from day 5
In the experiments described above, the ECM coating of plates was produced by confluent layers of mesenchymal cells whose concentration was approximately 40-fold greater than the highest concentration of mesenchymal cells used in the co-cultures. To clarify whether the strong stimulation of neural stem/early progenitor cell proliferation and differentiation in co-cultures was caused by the presence of mesenchymal ECM in co-cultures, cortical cells were mixed with decreasing numbers of MSC or SB623 cells and co-culture was conducted under non-adherent conditions, in plates coated with poly-D-lysine (PDL). As controls, cortical cells were plated alone, with or without FGF2 and EGF; and mesenchymal cells were plated alone at the highest concentration used in co-culture.
For poly-D-lysine (PDL) coating, plates were coated with PDL (Sigma-Aldrich) at 10 μg/ml in water for 1 hour at room temperature. Then the PDL solution was aspirated, wells were allowed to dry, and then washed once with PBS. Before cells were plated in these coated wells, the PBS was replaced with a portion of the neural growth medium, and the plates were warmed in an incubator during cell suspensions preparation.
Cells were co-cultured for 14 days. During this time, cell aggregates were formed in all wells. In cultures of mesenchymal cells alone, these aggregates were small and no visible increase in their size was observed during the culture period; indeed, many of them died. Under all other conditions, aggregates grew significantly, forming typical neurospheres. At the end of the incubation, the total contents of all wells was collected, pelleted, and lysed in equal volumes of lysis buffer; then tested by qRT-PCR for the expression of rat neural markers.
When neurospheres observed in co-cultures containing the highest dose of MSC were compared to neurospheres grown in the presence of FGF2 and EGF, the former had one-third the level of Nestin gene expression, but 2.5-fold higher Dcx expression and 55-fold higher GFAP expression, as well as higher levels of MAP2, CNPase, and rGAP expression (1.5, 3.5, and 3 times, respectively). Human GAP (huGAP) mRNA was detected in MSC cultured alone under non-adherent conditions, but its levels were dramatically reduced when the same number of cells were co-cultured with neural cells (17.2 and 3.1, respectively). Lower doses of MSC exhibited huGAP expression levels that were below quantification limits. This indicated that the neural cell environment, in combination with non-adherent conditions, was unfavorable for growth of mesenchymal cells, and they did not survive at lower plating doses. Nevertheless, their ability to stimulate neural development persisted.
In cortical cells cultured in the absence of bFGF and EGF, gene expression of all neural markers was low, as was expression of rat GAP. In co-cultures with MSC, expression of all markers was increased in a dose-dependent fashion, despite the fact that the majority of MSC died in co-cultures, as well as when cultured alone (as indicated by huGAP levels).
Similar results were obtained using SB623 cells instead of MSC. These results show that the stimulatory effects of mesenchymal cells observed in co-cultures on ECM are not due to the ECM itself.
The effects of attachment conditions on the differentiation of neural cells in co-culture were assessed. To this end, neural cells were co-cultured with MSC on SB623 cell derived ECM-coated plates, on ornithine/fibronectin-coated plates, and on Ultra Low Attachment (ULA) plates (Corning, Lowell, Mass.). Ornithine/fibronectin-coated plates supported attachment of MSC. On ULA plates, neural cells were cultured either with a five-fold higher concentration of MSC (compared to co-culture on ECM or ornithine/fibronectin) or with 20 ng/ml each of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF).
Ornithine/fibronectin coating (Orn/FN) was prepared by incubating wells with 15 ug/ml poly-L-ornithine (Sigma-Aldrich) in PBS, overnight at 37° C., then washing the wells 3 times, followed by incubation overnight with PBS at 37° C. After this, wells were incubated with 1 ug/ml bovine fibronectin (Sigma-Aldrich) in PBS, for 3-30 hours and washed once before plating cells.
A mixed suspension of rat cortical cells and human MSC (neural cells/MSC ratio 10:1, 1.5×104/cm2) was plated on SB623 cell ECM-coated plates and on ornithine/fibronectin-coated plates. Neural cells mixed with either a five-fold higher concentration of MSC or with FGF2 and EGF (as described above) were plated on ULA plates. Marker expression was assayed by qRT-PCR at either 5 or 7 days of co-culture.
The results are shown in
Non-attached growth (on ULA plates) had a detrimental effect on growth and survival of MSC, as evidenced by the fact that huGAP levels were very low despite a 5-fold greater MSC concentration on ULA plates compared to ECM and orn/FN-coated plates. Under non-adherent conditions, FGF2/EGF and MSC supported similar levels of Nes and CNPase expression.
In summary, co-cultures of neural cells and MSC grown on ECM-coated plates contained the most diverse neural cell population, in contrast to co-cultures on Orn/FN or in ULA wells. In particular, co-culture on ECM supported levels of nestin expression that were comparable to those in FGF2/EGF-driven spheroids, and also supported the highest levels of GFAP expression under any of the conditions tested.
In light of the positive effects of ECM on the abundance of nestin-expressing cells, as described in the preceding example, the effect of the heparan sulfate proteoglycan components of the ECM on nestin expression were investigated.
For these experiments, plates were coated with SB623 ECM as described supra, then ECM was treated with a solution of Heparinase 1 (Sigma-Aldrich) in 10 mM HEPES, pH 7.4, 100 mM NaCl, and 4 mM CaCl2 overnight at room temperature and washed once. Heparinase concentrations are given in the legend to
Rat cortical cells were cultured on the plates containing heparinase-treated ECM. After five days of culture, nestin expression was assayed by qRT-PCR. The results, shown in
Quantitative RT-PCR was used to measure the expression, by MSC, of mRNA encoding certain growth factors and cytokines, as shown in Table 4 below.
A number of growth factors and cytokines produced by MSC were tested for their ability to stimulate neurogenesis and gliogenesis, by adding the recombinant factor, either by itself or with 5% MSC conditioned medium (MSC-CM), to neural cells cultured on ECM.
ECM was produced by growing SB623 cells in culture, then washing the cells from the culture vessel. Primary embryonic rat cortical cells (Brain Bits, Springfield, Ill.) were cultured on the ECM in the presence of 5% MSC conditioned medium and a particular recombinant cytokine or growth factor, as shown in Table 5 below, for 5 or 7 days. The cells were then assayed for the expression of various rat neuronal and glial markers by species-specific quantitative RT-PCR. A summary of these results is shown in Table 5.
Quantitative results showing the effects of three factors (EGF, BMP6 and HB-EGF) on expression of markers for neuronal precursors (Nestin), nascent neurons (DCX), oligodendrocytes (CNPase) and astrocytes (GFAP) are shown in
Fibroblast growth factor-2 (FGF2) was secreted by MSC (Table 4, supra) and addition of FGF2 to cortical cells stimulated expression of nestin, MAP2 and CNPase (Table 5, supra). Two additional experiments were conducted to confirm the role of FGF2 in stimulating nestin expression. In the first, a blocking antibody to FGF2 was added to co-cultures of neural cells and MSC. In the second, MSC conditioned medium was depleted of FGF2 and added to cultures of neural cells.
For the first experiment, two antibodies were used: bFM1 (a FGF2 neutralizing antibody that recognizes both rat and human FGF2) and bFM2 (a FGF2-specific non-neutralizing antibody), both obtained from Millipore, Billerica, Mass. Rat cortical cells were cultured at a concentration of 5,000 cells/well in the presence or absence of MSC at a concentration of 200 cells/well. The antibodies were added to co-cultures of cortical cells and MSC at a concentration of 0.2 ug/ml.
The results of this analysis are shown in
For the second experiment, FGF2-depleted MSC-CM, and control MSC-CM, were prepared as follows. MSC-CM was incubated with the anti-FGF2 neutralizing antibody bFM1, or with control mouse IgG1, at 5 ug/ml overnight at 4° C. on a rotisserie shaker, followed by the addition of protein A/G-plus Agarose (Santa Cruz Biotechnology, Santa Cruz, Calif.) and incubation for 1 hour. After removal of the beads by centrifugation, the supernatant was collected and sterile-filtered.
Neural cells were cultured on ECM-coated plates with no further additions, or with addition of MSC-CM, FGF2-depleted MSC-CM, or control immunoprecipitated MSC-CM, for 5 days, and nestin mRNA expression was measured by qRT-PCR. The results are shown in
These results indicate that MSC-derived FGF2 is a primary factor responsible for nestin induction in neural cells, and also indicate that basal nestin levels in cortical ECM-based cultures were dependent on FGF2, either of rat or human origin.
The effect of mesenchymal stem cells was compared with the effect of conditioned medium from mesenchymal stem cells on the expression of nestin and GFAP in neural cell cultures on ECM-coated plates. As shown in
Bone morphogenetic protein-4 (BMP4), a factor secreted by MSC (Table 4, supra), stimulated expression of GFAP when added to cultures of neural cells (Table 5, supra). To confirm the role of BMP4 in astrogenesis, co-culture of rat neural cells and MSC was conducted in the presence of a BMP agonist (noggin) and in the presence of an anti-BMP4 antibody. Recombinant human Noggin protein, obtained from R&D Systems (Minneapolis, Minn.), was included in the co-cultures at a final concentration of 30 ng/ml. Polyclonal goat anti-BMP4 and normal goat IgG control were used in co-cultures at 2 ug/ml. These reagents, as well as mouse IgG1 isotype control were obtained from R&D Systems (Minneapolis, Minn.).
Attempts were made to immunoprecipitate BMP4 from MSC conditioned medium by incubating MSC-CM with the anti-BMP4 antibody or control (goat IgG, or no antibody) at 5 ug/ml overnight at 4° C. on a rotisserie shaker following by the addition of protein A/G-plus Agarose (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 1 hour. After removing beads by centrifugation, the supernatant was collected and sterile-filtered. However, GFAP levels did not differ significantly in neural cells cultured in the presence of MSC-CM, compared to neural cells cultured in the presence of BMP4-depleted MSC-CM. Nonetheless, the anti-BMP4 antibody was capable of blocking GFAP induction driven by recombinant BMP4.
The lower astrogenic activity of MSC-CM compared to MSC; the partial decrease of GFAP levels in co-cultures treated with an anti-BMP4 neutralizing antibody; and the lack of effect of BMP4 immunodepletion on the astrogenic activity of MSC-CM, taken together, suggest either that the active BMP4 astrocyte-inducing activity resides within a cell-ECM compartment (rather than in the medium), or that it is produced by rat cells.
To test whether active BMP4 in co-cultures was produced by MSC or by the rat neural cells, production of BMP4 by the MSC was inhibited, prior to co-culturing, using siRNA. For siRNA transfection, freshly thawed MSC were plated at 0.4×106 cells per 6-well plate in αMEM/10% FBS. Next day cells were transfected with either ON-TARGETplusSMARTpool human BMP4 siRNA or a control non-targeting pool at 25 nM, using DharmaFECT®1 (all reagents from Thermo Scientific Dharmacon®, Lafayette, Colo.) according to the manufacturer's instructions. Next day cells were trypsinized and lifted, and trypsin was inhibited by adding FBS. Cells then were washed twice in Neurobasal medium and counted (viability was usually greater than 95%).
The day after transfection, equal cell numbers of transfectants were plated with rat cortical cells and co-cultured for 5 days. On day 5, rat GFAP mRNA levels and human BMP4 levels were assayed. The results, shown in
On ECM, the growth of Nes+ cells was significantly augmented in a dose-dependent fashion by live mesenchymal cells or their conditioned medium, as demonstrated using immunostaining and qRT-PCR, while on PDL the response to these factors was reduced (
Mesenchymal stromal cells in co-cultures promoted both neuritogenesis and de novo neuron formation from Nes+ cells (
Mesenchymal cells, including MSC and SB623, increased the formation of new neurons on ECM, as demonstrated by a massive appearance of double-positive Nes+MAP2+ cells around day 7 in the co-cultures (shown on
At the plating densities used here, GFAP expression was a hallmark of ECM-based cultures and was absent in PDL-based cultures. GFAP protein staining was closely associated with the staining for Nestin filaments in Nes+ filamentous or flat stellar-shaped cells around day 7 (
Due to the lack of GFAP+ cell growth on PDL, it is not clear whether the soluble short-lived astrocyte-inducing factors required ECM for their signaling, or if the immature, round Nes+ cells simply did not express receptors for the inducing factors. Indeed, mesenchymal cell-derived factors TGFβ, HGF, and BMPs were implicated in promoting astrogenesis [26, 43-45]; all these factors are ECM-bound in their inactive form and have short life span when released from ECM. Another illustration of the significance of mesenchymal cell-derived soluble and insoluble factors for astroglial differentiation comes from the observation of non-adherent cultures (
On ECM, astrocytes-like Nes+ cells that did not express GFAP were observed (
Oligodendrocytic differentiation was monitored using an early oligodendrocytic marker, the myelin-processing enzyme CNPase, whose expression typically follows O4 expression and precedes the expression of myelin basic protein (MBP) [49]. In the experiments described herein, appearance of CNPase protein was detected relatively late after the appearance of its mRNA, but was expedited by the presence of MSC or SB623 cells (
These results indicate the existence of a cell density-dependent inhibition of oligodendrocyte differentiation; however, it is unclear whether mesenchymal cells are responsible directly or indirectly. Expansion and differentiation of oligodendrocyte precursors are controlled by cell density [50, 51] and, although the control mechanism is unknown, it has been reported that local cell-to-cell interactions, rather than long range diffusible factors, were implicated; and that the effect is cell type-specific, i.e. it was mediated specifically by oligodendrocytic lineage [50]. The results of the assays described herein are consistent with the possibility that higher doses of mesenchymal cells inhibit oligodendrocyte differentiation indirectly, by increasing the proliferation of early oligodendrocyte precursors. On ECM, MSC-CM induced more than 3-fold higher CNPase expression than did live cells (Table 2), and much less GFAP expression was detected under these conditions. These observations suggest an interplay between, and balancing of, rates of proliferation and differentiation for astrocytes compared to oligodendrocytes. The data disclosed herein also supports the notion that ECM itself can play a role in promoting oligodendrocyte proliferation and differentiation [52]. Indeed, on PDL, even in the presence of MSC or MSC-CM, CNPase expression levels were low and the protein was not detected over the course of 2 weeks, while on ECM the protein was detected, even in the absence of other additives.
The methods and compositions disclosed herein enable the quantitative analysis of neuropoietic activity of test substances in mixed cross-species co-cultures. In this system, mesenchymal cell-derived ECM is used as a substrate for adherent co-culturing; neural cells are cultured in the same microenvironment from start to finish, without external growth factors; primary neural cells and test substances (e.g., MSC preparations) are co-cultured directly, at low cell plating density. The system allows analysis of secreted, diffusible, cell-associated and matrix-associated factors. Analysis can be conducted in a microplate format; using qRT-PCR-based readout for neural markers from total lysates.
Mesenchymal cell-derived ECM was chosen as a substrate for neural cell co-cultures based on previous observations that human MSC-derived ECM and, to a greater extent, SB623 cell-derived ECM, permit the growth of rat embryonic cortical cells and their subsequent differentiation to neuronal and glial lineages at relatively low cell plating densities and in the absence of growth factors [28]. Herein it is disclosed ECM coating created a favorable environment for Nestin-positive cell growth. The integral heparan sulfate proteoglycans (HSPG) of the ECM were important, since Heparinase 1 pre-treatment of ECM diminished nestin levels in cultures, in contrast to control-treated wells. The role of HSPGs suggested involvement of FGF2 signaling. Indeed, an antibody blocking FGF2, though not a control antibody, decreased nestin expression below basal levels in neural cultures. This suggests that FGF2 plays an important role in ECM-based cultures. FGF2 (of rat or human origin, or both) can provide physiological stimulation that supports the survival and the slow proliferation of neural stem/early precursor cells and enables the subsequent differentiation of the adherent culture. Recent reports identified mesenchymal cell-derived ECM as an integral part of an in vivo neural stem cell niche in the form of extravascular basal laminae (fractones) and its HSPGs were implicated in the accumulation of FGF2 [31, 32]. This observation justifies the use of mesenchymal ECM substrate for neural cell culturing to model a stem cell niche. Although most of the results disclosed herein were obtained using SB623-cell-derived ECM, MSC-ECM-based systems also produce similar results, although at longer culturing times.
When a cortical cell population is grown on ECM in the absence of growth factors or other test substances, differentiated glial cells are detected in 2-3 weeks [28]. In the presence of MSC, the neural population proliferated and differentiated significantly more rapidly, in an MSC-dose dependent manner (see Examples). The species-specific qRT-PCR readout method described herein is capable of detecting induction of rat neural markers in the presence of as little as 50 human MSC per 5000 rat neural cells. Levels of neural marker expression reflected a cumulative outcome of several processes in co-cultures. For example, an MSC-driven increase in total nestin expression (
MSC are known to secrete many growth factors that have been shown to participate in the maintenance of neural precursors and neurodifferentiation in vivo [reviewed in 35] and the secretion of some of them, including BMP4, FGF2, EGF, VEGF, and PDGF-AA has been confirmed in some MSC batches used here (30 and co-owned US Patent Application Publication No. 2010/0266554). Blocking experiments demonstrated that MSC-produced FGF2 was the major factor responsible for MSC-driven nestin induction in co-cultures (Example 13). Nestin-inducing activity could also be efficiently transferred by MSC-CM; and approximately 85-90% of it could be removed from MSC-CM by immunoprecipitation of FGF2. The crucial role of FGF2 in the maintenance of neural stem cell is well known (reviewed in 36, 37); but the results described herein demonstrate that the contribution of MSC-derived FGF2 to MSC-driven nestin induction overwhelmed other possible contributors.
The induction of astrogenesis (measured as GFAP expression) under serum-free conditions is a hallmark of ECM-based cultures of E18 cortical cells. GFAP protein staining was closely associated with the staining for nestin filaments around day 7 (Example 2). Moreover, the formation of nestin filaments accompanying Nes+ cell spreading seemed to be a pre-requisite for astrocytic differentiation, since no GFAP induction was observed on other substrates that did not support Nes+-cell spreading (38). MSC greatly promoted GFAP expression. BMPs were found herein to be major mediators of this effect, since Noggin, a negative regulator of BMP activity, inhibited ˜90% of GFAP induction in co-cultures. BMP4 was abundantly expressed in MSC (39 and Table 4). An antibody that blocks human BMP4 eliminated ˜60% of GFAP induction, indicating that human BMP4 was a major astrogenic BMP. BMP4 was previously implicated in mediating astrogenic effects of specially induced rat MSC in co-cultures with mouse neurospheres (40). Example 14 shows that MSC-CM was less astrogenic than MSC, in agreement with a previous report (25). The residual astrogenic activity of MSC-CM could not be removed by immunodepleting BMP4; this could mean that BMP4 was not responsible for the residual astrogenic activity, or that it was present in an inactive form. However, MSC transfected with BMP4-siRNA, but not with control siRNA, when plated in co-cultures, showed reduced astrogenic activity, while FGF2 expression was not altered by the transfection (Example 14). Taken together, these results suggest that astrogenic activity of MSC is mediated in part by BMPs (specifically, human BMP4) and that the active BMP4 was either cell-associated or bound to the ECM, and not secreted into the medium. These results do not exclude the possibility that other MSC-derived factors, such as TGFβ, are involved (25, 42), although blocking TGFβ1 in co-cultures did not result in inhibition of astrogenesis.
Oligodendrocytic differentiation was monitored using an early oligodendrocytic marker, the myelin-processing enzyme CNPase, whose expression typically follows O4 expression, precedes the expression of myelin basic protein, and increases throughout the maturation process (43). In agreement with previous reports (24, 44, 45), oligodendrogenesis in the co-cultures described herein was clearly MSC-dependent; however, the timing of MSC-dose response was different from that of astrogenesis. On day 5 of co-culture, there was a direct relationship between MSC dose and levels of CNPase expression; whereas, on day 7, CNPase activation by increasing doses of MSC reached a plateau, beyond which further increases in MSC dose resulted in lower levels of activation. (
Disclosed herein is an in vitro system that enables the quantitative multifactorial analysis of the effects of a test substance on a primary neural cell population. The system preserves the complexity and some of the intrinsic interactions of a primary cell population. This system can be used to identify and quantitate soluble, cell-associated and/or matrix-bound neuropoietic factors and has been used to show the importance of soluble FGF2 and cell-associated or matrix-bound BMP4 in neuronal and astrocyte development, respectively. Finally, the system can be used for comparing the potencies of various lots of MSC or their derivatives (e.g., SB623 cells), as well as for studying the effects of neural population on MSC.
The data presented herein suggest that SB623 cells induce the proliferation and differentiation of early neural precursors more efficiently than do their parental MSC.
In summary, the inventors have described an in vitro system that enables the imaging and the high-throughput quantitation of the effects of substances (such as, for example, MSC, SB623 cells and their products) on various stages of neural cell growth and differentiation. This system will facilitate the study of a number of differentiative processes, including, for example, MSC/neural cell interactions, and serve as a basis for potency assays for neuroregenerative cell-based therapies.
In addition, neurogenic effects of FGF2, and astrocytogenic effects of BMP4, have been demonstrated. Accordingly, FGF2 and BMP4 can be substituted for the neural precursor cells or for any of the neural cells described in co-owned U.S. Pat. No. 7,682,825, for use in treatment of a disease, disorder or condition of the central or peripheral nervous system. To that end, the disclosure of U.S. Pat. No. 7,682,825 is incorporated by reference herein, in its entirety. Furthermore, FGF2 and BMP4 can be substituted for the neuronal precursor cells, the MASC-derived neuronal cells, or any of the graft-forming units described in co-owned U.S. Pat. No. 8,092,792, for use in treatment of a central nervous system lesions (e.g., ischemic stroke, hemorrhagic stroke). To that end, the disclosure of U.S. Pat. No. 8,092,792 is incorporated by reference herein, in its entirety.
This application is a continuation of U.S. patent application Ser. No. 13/589,849 filed Aug. 20, 2012 which claims the benefit of U.S. Provisional Patent Application No. 61/575,378 filed Aug. 19, 2011 and U.S. Provisional Patent Application No. 61/580,991 filed Dec. 28, 2011, the contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61575378 | Aug 2011 | US | |
| 61580991 | Dec 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13589849 | Aug 2012 | US |
| Child | 16406937 | US |
