[0028] In another aspect of the invention, a method for the expansion or growth of stem cells is provided, by incubating at least a portion of a
placenta in a
growth medium to condition the medium, and contacting at least one stem cell with the
growth medium hemochorial, epitheliochorial, or endotheliochorial. In a preferred embodiment the
placenta is hemochorial. The
placenta may be collected subsequent to
vaginal delivery or collected pre-term by cesarean section, depending on biological properties desired. In a preferred embodiment the placenta is hemochorial. The stem cell can be, for example, a
mesenchymal stem cell, or a fetal stem cell. The stem cells can be derived from an
umbilical cord, such as, for example, from
umbilical cord blood. The stem cells can be derived from an
umbilical cord that expresses a CD34+
cell marker. The umbilical cord stem cells and said placenta can be derived, for example, from a
mammal, such as a human. The growth medium can also contain, if desired, a
growth factor, combinations of growth factors, or substantial
nutrient content allowing for increased viability of the stem cells. The incubating step can occur, for example, at a temperature range of from about 32° C. to about 40° C. The placenta can be removed from the medium prior to the contacting step, if desired. The placenta can either be perfused with the medium or it may be cultured in the medium at conditions that allow for release of growth factors.
[0034] Another embodiment is the use of LPCM alone or in combination with other approaches expanding cells that have been generated for a specific
phenotype, and are at risk of losing the
phenotype that was artificially endowed upon them. Specifically, it is known that administration of a certain compounds to stem cells induces differentiation into certain lineage-specific progenitors. For example, addition of
thrombopoietin alone or in combination with
interleukin 11 to early hematopoietic stem cells will promote the preferential production of megakaryocytic progenitors. One embodiment of the current invention is the ability of LPCM, alone or in combination with other growth factors and / or culture conditions to maintain and expand the new
phenotype of the differentiated
progenitor cell without stimulation of terminal differentiation. For example, subsequent to increasing the numbers of megakaryocytic progenitors in a
stem cell culture, LPCM may be added to maintain said progenitors and expand their numbers.
[0037] Another embodiment of the invention is a stimulator of proliferation of
totipotent stem cells such as such as human embryonic stem cells characterized by expression of markers such as SSEA-4, GCTM-2
antigen, TRA 1-60, Cripto,
gastrin-releasing
peptide (GRP)
receptor,
podocalyxin-like
protein (PODXL), or
human telomerase reverse transcriptase (hTERT). The LPCM can be used as a stimulator of proliferation alone or as an additive to media known to be useful for culturing said cells. An example of such a
tissue culture media is Dulbecco's modified
Eagle's medium (DMEM). In an ideal embodiment LPCM is used in such a manner and under such conditions so as to alleviate the need for serum or feeder cells in the culture of human embryonic stem cells.
[0062] Within the embodiments of the invention is the use of LPCM, or extracts thereof, to enhance proliferation of stem cells within a living
organism. Administration of such media can be performed systemically, or in a localized environment. Clinical situations where administration of such placentally conditioned media is desirable can include conditions where an increase in the number of stem cells is sought due to
disease or
senescence of endogenous stem cells. Specific aspects of this include conditions in which a higher number and / or more rapid
recovery of stem cells is needed after a
medical procedure. One such situation would be post bone marrow transplant where expansion of hematopoietic cells is desirable in order for the patient not to succumb to bacterial or viral infections. Specifically, LPCM may be used in conjunction with a
growth factor that stimulates preferential differentiation of the
bone marrow stem cell into the granulocytic and / or monocytic lineage such as G-CSM or GM-CSF. Such an expansion of granulocytic and monocytic precursors would be useful in enhancing immunological defenses subsequent to a bone marrow transplant. If clinically desirable the number of endogenous dendritic cells can also be expanded through administration of cytokines such as flt-3L in combination with LPCM. Accordingly, this invention provides methods and compositions that can be administered to a patient having undergone a bone marrow transplant that will enhance proliferation and bone marrow take.
[0076] In some embodiments of the present invention, a method for the expansion or growth of stem cells without substantially inducing differentiation is provided, by incubating at least a portion of a placenta in a growth medium to condition the medium, and contacting at least one stem cell with the growth medium. The placenta can be derived from a
mammal. The placenta can be derived from a human. The placenta can be derived preterm. The placenta can be derived at term. The placenta can be perfused for a period of time with a
cell culture media. The
cell culture media can be supplemented, for example, with a single or a plurality of growth factors. The growth factors can be selected from, for example, a WNT signaling
agonist, TGF-b, bFGF, IL-6, SCF, BMP-2,
thrombopoietin, EPO, IGF-1, IL-11, IL-5, Flt-3 / Flk-2 ligand,
fibronectin, LIF, HGF, NFG,
angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre, or a mixture thereof. The media can be capable of maintaining viability of a substantial portion of the
placental tissue during the
perfusion process. The media can be selected, for example, from Roswell Park Memorial Institute (RPMI-1640), Dublecco's Modified Essential Media (DMEM),
Eagle's Modified Essential Media (EMEM), Optimem, and Iscove's Media. The source of serum can be added to the media. The concentration of serum in the media can be approximately between 0.1% to 25%. The concentration of serum in the media can be approximately 10%. The serum can be selected from adult human serum, fetal human serum, fetal calf serum and umbilical
cord blood serum. The contacting step can occur after the incubating step. The contacting step can occur simultaneously with the incubating step. The incubating step can occur from about 1 second to about 3 weeks. The incubating step can occur from about 24 hours to about 10 days. The contacting step can occur from about 1 second to about 3 weeks. The contacting step can occur from about 24 hours to about 10 days. The placenta can be, for example, a hemochorial, epitheliochorial, or endotheliochorial placenta. The
perfusion can be accomplished, for example, through the use of a
perfusion apparatus cannulated to blood vessels connected to the placental body. The perfusion apparatus can allow for control of intravasular pressure,
oxygen content,
carbon dioxide content, pH, and flow rate of the perfused media flowing through the
placental blood vessels. The intravasular pressure of the perfusate can be maintained, for example, at 30-80 Hg. The intravasular pressure of the perfusate can be maintained at 60 Hg.
[0083] In an additional embodiment of the present invention, a method of culturing a placenta in its original 3-dimensional structure is provided, in such a manner as to reproduce the
in vivo environment in which it resides in the pregnant woman, thus retaining capability of generation and
secretion of growth factors and proteins that maintain the fetal regenerative capacity. The method involves acquiring a placenta under sterile conditions, cannulating blood vessels of the placenta in order to allow proper perfusion in circumstances similar to as if the placenta was performing its
in vivo functions, perfusing the placenta with a
nutrient mix in a buffer that would mimic physiological conditions, maintaining a temperature and physical environment similar to that found in the pregnant woman's body, and imitating conditions of flow, pH,
oxygenation, and pressure similar to that found in the body. The perfusion of both the maternal and fetal circulatory components of the placenta can be performed. A
nutrient mixture can be used that possesses similar nutrient requirements as the fetal and maternal circulation, respectively. A temperature of 37° C. can be maintained during the perfusion process. The pH can be monitored, for example, by the perfusion apparatus in a real-time basis, and adjusted using adequate quantities of acids, bases, or buffers. The
oxygen content can be maintained similar to that found in the fetal and maternal circulatory contribution to the placenta. The
oxygen content may be increased, for example, through the use of adding natural or artificial oxygen carriers to the perfusion solution. An
oxygenator may be attached to the perfusion apparatus, in conjunction with, or separately, from an
oxygen sensor, the combination being used to adjust in real-time
oxygen content. The osmolality can be maintained, for example, through the use of known means such as addition of
albumin or colloids to the perfusion solution.
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