STEM CELL TECHNOLOGY DA-1

ARARSO SHEMENI KABETO

REG NO:- 18BBT0264

FACULTY :- Dr TAMIZHSELVI R

Submission Date 26/07/2020

Title: Explain the culture medium required for regulating the differentiation of
stem cells into specific lineages.
  The medium used for the diff of msc to osteoblast

 The osteogenic induction medium

containing dexamethasone (Dex), β-glycerophosphate and ascorbic acid, has
been generally used to induce the MSC differentiation into osteoblast, by
increasing the alkaline phosphatase activity and accelerating bone
mineralization.
TM
1.) Lonza's hMSC Osteogenic Differentiation BulletKit Medium

Stem cells possess the unique ability to differentiate into multiple lineages of cells including
osteoblasts. Lonza’s hMSC Osteogenic Differentiation BulletKitTM Medium is a serum
containing medium designed to induce osteogenic differentiation of human bone marrow derived
mesenchymal stem cells (hMSC) into mature, functionally active osteoblasts in as few as 14
days. Differentiation can then be validated by a variety of methods including the use of Lonza’s
OsteoImageTM Bone Mineralization Reagent which provides a quantitative value related to the
amount of osteogenic differentiation.
Partial List of Cells Differentiated with hMSC Osteogenic Differentiation BulletKitTM Medium*:

 Human bone marrow derived stem cells

 Human umbilical cord blood derived stem cells
 Human umbilical cord matrix derived stem cells
 C57BL/6 mice bone marrow derived stem cells
 Human adipose derived stem cells
 Human periodontal ligament derived stem cells
 Human hamstring tendon derived stem cells

The hMSC Osteogenic Differentiation BulletKitTM Medium provides a differentiation medium

guaranteed to induce osteogenic differentiation of bone marrow derived hMSCs into mature,
functionally active osteoblasts; Each bottle of the hMSC Osteogenic Differentiation
BulletKitTM Medium contains enough differentiation medium to fully differentiate up to 750,000
bone marrow derived hMSCs [approximately 25 wells in a 6-well plate format] into mature,
functionally active osteoblasts.
The hMSC Osteogenic Differentiation Medium is offered as a BulletKitTM Medium (catalog no.
PT-3002) which includes both the basal media and the necessary supplements for osteogenic
differentiation of bone marrow derived hMSCs.
Lonza guarantees the performance of cells only if appropriate media and reagents are used
exclusively and the recommended storage and use protocols are followed. Any modifications
made to the recommended cell systems including the use of alternative media, reagents or
protocols, will void cell and media performance guarantees.
  Content
1 x hMSC Osteogenic Differentiation Basal Medium (PT-3924), 170 mL
1 x hMSC Osteogenic Differentiation SingleQuotsTM Supplements Kit (PT-4120)

2.)MesenCult™ Osteogenic Differentiation Kit (Human) medium

MesenCult™ Osteogenic Differentiation Kit (Human) is specifically formulated for the in
vitro differentiation of human mesenchymal stem and progenitor cells (MSCs) into cells
of the osteogenic lineage. This kit is suitable for the differentiation of human bone
marrow (BM)- derived MSCs previously culture-expanded in serum-containing medium
(e.g. MesenCult™ Proliferation Kit [Human; Catalog #05411] or MesenCult™-hPL
[Human; Catalog #05439]) or serum- and animal component-free MesenCult™-ACF Plus
Medium (Catalog #05445). NOTE: Complete MesenCult™ Osteogenic Differentiation
Medium must be supplemented with L-Glutamine (Catalog #07100).

Preparation of Complete Osteogenic Differentiation Medium (Human)

 Use sterile techniques to prepare complete Osteogenic Differentiation Medium
(Basal Medium + 5X Supplement + L-Glutamine). The following example is for
preparing 50 mL of complete medium. If preparing other volumes, adjust
accordingly. NOTE: For aliquoting and storing either the supplement or the
complete medium, polypropylene tubes are strongly recommended (e.g.
Falcon® Conical Tubes, 15 mL [Catalog #38009]). Thaw the 5X Supplement at
room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly. NOTE:
Once thawed, use immediately or aliquot and store at -20°C. Do not exceed the
shelf life of the supplement. After thawing the aliquoted supplement, use
immediately. Do not re-freeze.
 Add 10 mL of 5X Supplement to 40 mL of Basal Medium. Mix thoroughly.
 Add 0.5 mL of L-Glutamine (200 mM; Catalog #07100) to reach a final
concentration of 2 mM. Mix thoroughly. NOTE: If not used immediately, store
complete Osteogenic Differentiation Medium at 2 - 8°C for up to 1 week.

Directions for Use

 Please read the entire protocol before proceeding. For instructions on culturing human
MSCs using the MesenCullt™ media listed below, refer to the Product Information
Sheets available at www.stemcell.com. • MesenCult™ Medium (Human; Catalog
#05411) • MesenCult™-hPL Medium (Human; Catalog #05439) • MesenCult™-ACF Plus
Medium (Catalog #05445) For differentiating to the osteogenic lineage, use culture-
expanded human MSCs between passage 1 - 4. The following protocol is for setting up
differentiation assays using human BM-derived MSCs in a 6-well plate. If using other
cultureware, adjust volumes accordingly.
NOTE: Only use tissue culture-treated cultureware.
 Plate cells in 2 mL of growth medium per well. Recommended cell plating densities
are as follows:
• MesenCult™ Medium (Catalog #05411): 8 - 16 x 10^3 cells/cm2
• MesenCult™-ACF Plus Medium or MesenCult™-hPL Medium (Human): 4 - 8 x 10^3
cells/cm2 NOTE: If using MesenCult™-ACF Plus Medium, ensure to coat cultureware
as described in the Product Information Sheet (Document #DX22329).

 Incubate at 37°C in 5% CO2 with ≥ 95% humidity until cells are approximately 90 -
98% confluent. This takes approximately 1 - 5 days depending on the expansion
medium used.
 Aspirate medium and replace with 2 mL of complete Osteogenic Differentiation
Medium per well. Incubate at 37°C in 5% CO2 with ≥ 95% humidity.
 Change medium every 3 - 4 days until bone matrix formation occurs (approximately
10 - 15 days).
 Visualize osteogenic differentiation by staining with Alizarin Red S, alkaline
phosphatase, or silver nitrate (von Kossa). NOTE: The level of osteogenic
differentiation for MSCs may vary depending on cell source, donor, and previous
culture conditions.
3.) Dulbecco's Modified Eagle Medium (DMEM)
Dulbecco's Modified Eagle Medium (DMEM) is one of the most widely used modification of
Eagle's medium. DMEM is a modification of Basal Medium Eagle (BME) that contains four fold
concentration of amino acids and vitamins. Additionally, the formulation also includes glycine,
serine and ferric nitrate. The original formulation contains 1000mgs/L of glucose and was
originally used to culture embryonic mouse cells. DMEM high glucose is a further modification
of original DMEM and contains 4500mgs glucose per litre. The additional glucose has proved to
be useful in cultivating various other cell lines including primary cultures of mouse and chicken
cells as well as various normal and transformed cell lines. AT068 is Dulbecco's Modified Eagle
Medium with 4.5gms glucose per litre and L-glutamine. It does not contain sodium pyruvate
and sodium phosphate. Users are advised to review the literature for recommendations
regarding medium supplementation and physiological growth requirements specific for
different cell lines.
  Directions :
 Suspend 13.3gms in 900ml tissue culture grade water with constant, gentle stirring until
the powder is completely dissolved. Do not heat the water.
 Add 3.7gms of sodium bicarbonate powder (TC230) or 49.3ml of 7.5% sodium
bicarbonate solution (TCL013) for 1litre of medium and stir until dissolved.
 Adjust the pH to 0.2-0.3 pH units below the desired pH using 1N HCl or 1N NaOH since
the pH tends to rise during filtration.
 Make up the final volume to 1000ml with tissue culture grade water.
 Sterilize the medium immediately by filtering through a sterile membrane filter with a
porosity of 0.22 micron or less, using positive pressure rather than vacuum to minimize
the loss of carbon dioxide.
 Aseptically add sterile supplements as required and dispense the desired amount of
sterile medium into sterile containers.
 Store liquid medium at 2-8°C and in dark till use.

Material required but not provided :

 Tissue culture grade water (TCL010)
 Sodium bicarbonate (TC230)
 Sodium bicarbonate solution 7.5% (TCL013)
 1N Hydrochloric acid (TCL003)
 1N Sodium hydroxide (TCL002)
 Foetal bovine serum (RM1112/RM10432)

Quality Control:

Appearance Off-white to Creamish white, homogenous powder.

 Solubility

Clear solution at 13.3gms/L.

 pH without Sodium Bicarbonate

6.80 -7.40

 pH with Sodium Bicarbonate

7.60 -8.20

 Osmolality without Sodium Bicarbonate

250.00 -290.00
  Osmolality with Sodium Bicarbonate

320.00 -360.00

Cultural Response
The growth promotion capacity of the medium is assessed qualitatively by analyzing the cells
for the morphology and quantitatively by estimating the cell counts and comparing it with a
control medium through minimum three subcultures. Endotoxin content NMT 5EU/ml Storage
and Shelf Life: 1. All the powdered media and prepared liquid culture media should be stored at
2-8°C. Use before the expiry date. In spite of above recommended storage condition, certain
powdered medium may show some signs of deterioration /degradation in certain instances.
This can be indicated by change in colour, change in appearance and presence of particulate
matter and haziness after dissolution. 2. Preparation of concentrated medium is not
recommended since free base amino acids and salt complexes having low solubility may
precipitate in concentrated medium. 3. pH and sodium bicarbonate concentration of the
prepared medium are critical factors affecting cell growth. This is also influenced by amount of
medium and volume of culture vessel used (surface to volume ratio). For example, in large
bottles, such as Roux bottles pH tends to rise perceptibly as significant volume of carbon
dioxide is released. Therefore, optimal conditions of pH, sodium bicarbonate concentration,
surface to volume ratio must be determined for each cell type. We recommend stringent
monitoring of pH. If needed, pH can be adjusted by using sterile 1N HCl or 1N NaOH or by
bubbling in carbon dioxide. 4. If required, supplements can be added to the medium prior to or
after filter sterilization observing sterility precautions. Shelf life of the medium will depend on
the nature of supplement added to the medium.

 The medium used for the diff of msc to chondrogenic

1.) PromoCell Mesenchymal Stem Cell Chondrogenic Differentiation

Media
This medium was developed for the directed differentiation of mesenchymal stem
cells (MSC) from bone marrow, the umbilical cord matrix (Wharton´s Jelly) and adipose
tissue into chondrogenic lineages.
 Mesenchymal Stem Cell Chondrogenic Differentiation Medium

 Cell culture medium for differentiation of mesenchymal cells into

chondrogenic lineages.
 PromoCell offers a complete Mesenchymal Stem Cell Media System including
growth media, differentiation media and human mesenchymal stem cells
(MSC).
 PromoCell offers five MSC Differentiation Media to efficiently induce
differentiation of MSC into adipogenic, chondrogenic (w/ and w/o inducers),
osteogenic or neurogenic lineages, respectively.
 PromoCell Mesenchymal Stem Cell Chondrogenic Differentiation Media was
developed for the directed differentiation of mesenchymal stem cells
(MSC) from bone marrow, the umbilical cord matrix (Wharton´s Jelly) and
adipose tissue into chondrogenic lineages.

2.) MesenCult™-ACF Chondrogenic

Differentiation Kit medium.
MesenCult™-ACF Chondrogenic Differentiation Medium is animal component-free (ACF) and
specifically formulated for the in vitro differentiation of human mesenchymal stromal cells
(MSCs; also known as mesencyhymal stem cells) into chondrogenic lineage cells, including
chondrocytes. This medium is suitable for the differentiation of human bone marrow (BM)-,
adipose tissue (AT)- and synovium (S)-derived MSCs previously culture-expanded in serum-
containing medium (e.g. MesenCult™ Proliferation Kit [Catalog #05411]) or animal component-
free MesenCult™-ACF Plus Medium [Catalog #05445]). MesenCult™-ACF Chondrogenic
Differentiation Medium induces robust chondrogenic differentiation of human MSCs with as few
as 3 x 10^5 cells and as early as day 14.

Advantages:
 Animal component-free (ACF) formulation
 Robust chondrogenic differentiation with as few as 3 x 10^5 MSCs and as early
as day 14.
 Strong expression of chondrogenic transcripts - Acan, Col2a, Sox9 and Col10a;
low expression of hypertrophic transcript Mmp13.
 Completes optimized ACF workflow for MSC isolation, expansion,
cryopreservation and chondrogenic differentiation

Components
 MesenCult™-ACF Chondrogenic Differentiation Basal Medium, 95 mL
 MesenCult™-ACF 20X Chondrogenic Differentiation Supplement, 5 mL
 3.) Human Mesenchymal Stem Cell (hMSC) Chondrogenic
Differentiation Medium BulletKitTM

Stem cells possess the unique ability to differentiate into multiple lineages of cells including
chondrocytes. Lonza’s hMSC Chondrogenic Differentiation BulletKitTM Medium is a serum
containing medium designed to induce chondrogenic differentiation, with the addition of TGF-
β3, of human bone marrow derived mesenchymal stem cells into mature, functionally active
chondrocytes in as few as 14 days. Differentiation can then be validated by a variety of methods
including staining with alcian blue or safranin.
Partial List of Cells Differentiated with hMSC Chondrogenic Differentiation
BulletKitTM Medium*:

 Human bone marrow derived stem cells

 Human umbilical cord blood derived stem cells
 Human umbilical cord matrix derived stem cells
 C57BL/6 mice bone marrow derived stem cells
 human adipose derived stem cells
 Human hamstring tendon derived stem cells

The hMSC Chondrogenic Differentiation BulletKitTM Medium, with the addition of TGF-β3
(sold separately) provides a differentiation medium guaranteed to induce chondrogenic
differentiation of bone marrow derived hMSCs into mature, functionally active chondrocytes;
Each bottle of the hMSC Chondrogenic Differentiation BulletKitTM Medium contains enough
differentiation medium to fully differentiate at least 2 million bone marrow derived hMSC
[approximately 4 complete 24-well plates] into mature, functionally active chondrocytes.
The hMSC Chondrogenic Differentiation Medium is offered as a BulletKitTM Medium (catalog
no. PT-3003) which includes both the basal media and the necessary supplements (with the
addition of TGF-β3 [sold separately, catalog no. PT-4124]) for chondrogenic differentiation of
bone marrow derived hMSCs. The basal medium (Differentiation Basal Medium -
Chondrogenic; catalog no. PT-3925) and the necessary supplements (HMSC SQ Kit - Chondro;
catalog no. PT-4121 and TGF Beta 3 for HMSC Chondro diff; catalog no. PT-4124) can also be
purchased individually.
Lonza guarantees the performance of cells only if appropriate media and reagents are used
exclusively and the recommended storage and use protocols are followed. Any modifications
made to the recommended cell systems including the use of alternative media, reagents or
protocols, will void cell and media performance guarantees
 Catalog #: PT-3003
 Culture system containing hMSC Chondrogenic Basal Medium (PT-3925)
and
 hMSC Chondrogenic SingleQuotsTM Kit Supplement (PT-4121) required
for hMSC chondrogenic differentiation.
  Content & Storage
Content
 1 x hMSC Chondrogenic Basal Medium (PT-3925)
 1 x hMSC Chondrogenic SingleQuot Kit (PT-4121)

4.) StemPro™ Chondrogenesis Differentiation Kit

medium.

The StemPro® Chondrogenesis Differentiation Kit has been developed for the
chondrogenic differentiation of mesenchymal stem cells (MSCs) in tissue-culture
vessels. The kit contains all reagents required for inducing MSCs to be committed to the
chondrogenesis pathway and generate chondrocytes. Using StemPro®
Chondrogenesis Differentiation Kit in combination with StemPro® MSC SFM or
MesenPRO RS™ Medium provides a standardized culture workflow solution for MSC
isolation, expansion, and differentiation into collagen matrix-producing chondrocytes.

Specifications
Cell Type: Stem Cells (Mesenchymal)

Classification: Serum-Free

Culture CO2
Environment:

Endotoxin Level: ≤50 EU/mL, ≤ 50 EU/ml

Form: Liquid

Glucose: Low Glucose

Glutamine: GlutaMAX™-I
HEPES Buffer: No HEPES

Phenol Red No Phenol Red

Indicator:

Purity or Quality Research Grade

Grade:

Research Differentiation
Category:

Sodium Sodium Bicarbonate

Bicarbonate Buffer:

Sodium Pyruvate Sodium Pyruvate

Additive:

Species: Human

Serum Level: Serum-Free

Product Line: Gibco®, StemPro®

 Contents & storage

This kit is shipped with a 100 ml basal medium and 10 ml growth supplement adequate
to formulate 100 ml of complete medium. Components require separate shipping and
storage requirements. The growth supplement is not sold individually.
StemPro® Osteocyte⁄Chondrocyte Differentiation Basal Medium 2 to 8°C (protect from
light)
StemPro® Chondrogenesis Supplement -5 to -20°C (in the dark).

The medium used for diff of msc to myogenic

 1.) Myogenic Differentiation medium
Myogenic differentiation is induced in amniotic fluid-derived progenitor cells by culture in
medium containing horse serum and chick embryo extract on a thin gel coat of
Matrigel . o initiate differentiation, the presence of 5-azacytidine in the medium for 24 h
is necessary. Phenotypically, the cells can be seen to organize themselves into bundles
that fuse to form multinucleated cells. These cells express sarcomeric tropomyosin and
desmin, both of which are not expressed in the original progenitor population.
The development profile of cells differentiating into myogenic lineages interestingly
mirrors a characteristic pattern of gene expression reflecting that seen with embryonic
muscle development . With this protocol, Myf6 is expressed on day 8 and suppressed
on day 16. MyoD expression is detectable at 8 days and suppressed at 16 days in
progenitor cells. Desmin expression is induced at 8 days and increases by 16 days in
progenitor cells cultured in myogenic medium.

 Myogenic differentiation potential of human tonsil-derived

mesenchymal stem cells and their potential for use to
promote skeletal muscle regeneration.
mesenchymal stem cells (MSCs), which have the ability to regulate the immune system and do
not form teratomas, may be isolated from various sources, including bone marrow , adipose
tissue, umbilical cord blood ,amniotic fluid, the placenta , dental pulp , the tonsils and urine .
For these reasons, MSCs have been recognized as a valuable source of cells which may be used
to propagate myogenic cells according to the protocols for differentiation. Several studies
examining the differentiation of MSCs into myogenic cells have been reported the use of MSCs
derived from adipose tissue, bone marrow, the placenta, amniotic fluid, umbilical cord blood
and urine .
The tonsils are a newly identified source of MSCs which may have potential therapeutic
applications. Tonsil-derived MSCs (T-MSCs) readily differentiate into cells of the mesodermal
lineage, including fat, cartilage and bone cells, and into cells of the endodermal lineage,
including hepatocytes .T-MSCs also exhibit similar immunosuppressive properties to bone
marrow-derived MSCs and adipose tissue-derived MSCs . As tonsillar tissues are discarded after
surgery, the isolation of stem cells from these discarded tissues is also a valuable means of
recycling human tissue for stem cell therapy.
  Skeletal Myogenic Differentiation of Mesenchymal Stem Cells
Isolated from Human Umbilical Cord Blood

Human umbilical cord blood (UCB) has been regarded as an alternative source for cell
transplantation and cell therapy because of its hematopoietic and nonhematopoietic
(mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells
(MSCs) are invariably present in UCB, several reports showed that MSC-like cells could be
consistently derived from human UCB and, moreover, could differentiate into various cells of a
mesodermal origin. However, it remains unclear whether these UCB-derived MSCs are also
capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human
UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion,
UCB-derived mononuclear cells gave rise to adherent layers of fibroblast-like cells expressing
MSC-related antigens such as SH2, SH3, alpha-smooth muscle actin, CD13, CD29, and CD49e.
More important, when these UCB-derived MSCs were incubated in promyogenic conditions for
up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation
pattern. Both flow cytometric and reverse transcriptase-polymerase reaction analyses showed
that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of
incubation but not after 2 weeks. At week 6, more than half of UCB-derived MSCs expressed
myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB-derived MSCs
possess a potential of skeletal myogenic differentiation and also imply that these cells could be a
suitable source for skeletal muscle repair and a useful tool of muscle-related tissue engineering.
etal bovine serum (FBS) was consistently used for the expansion. Moreover, while human
adipose-(ADSC) and umbilical cord-derived (ucMSCs) MSCs were shown to be capable of
differentiating into a skeletal myogenic phenotype using horse serum (HS) following
expansion in FBS-containing media , human bmMSCs have only been shown to differentiate
into skeletal muscle lineage cells in vitro using a mixture of cytokines including basic
fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), forskolin, and
neuregulin.