Stem Cell and Biomaterials Engineering Module 1

original name for Alamar Blue, (OBrien, 2000)

Resazurin

was used to measure the absorbance of Alamar Blue, resazurin and resorufin (OBrien, 2000)

DU7000 spectrophotometer (Beckman)

DU7000 spectrophotometer (Beckman) (OBrien, 2000)

was used to measure the absorbance of Alamar Blue, resazurin and resorufin

pink and highly fluorescent (OBrien, 2000)

resorufin

blue and nonfluorescent (OBrien, 2000)

resazurin

uncoloured and nonfluorescent (OBrien, 2000)

hydroresorufin

hydroresorufin fluorescence (OBrien, 2000)

uncoloured and nonfluorescent

resorufin fluorescence (OBrien, 2000)

pink and highly fluorescent

resazurin fluorescence (OBrien, 2000)

blue and nonfluorescent

resazurin reduction test (OBrien, 2000)

used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality

human neural stem cells (hNSCs), have two defining properties (Donato, 2007)

self-renewal and multipotentiality

preD abbreviation (Donato, 2007)

preaggregate differentiation protocol

ReNcell VM originated from (Donato, 2007)

bulk-derived culture

ReNcell CX originated from (Donato, 2007)

clonal cell line

ReNcell VM immortalized (Donato, 2007)

v-myc oncogene

ReNcell CX immortalized (Donato, 2007)

c-myc oncogene

Neurotoxicity (Joshi, 2020)

damage done to the nervous system when the brain is exposed to something toxic

damage done to the nervous system when the brain is exposed to something toxic (Joshi, 2020)

Neurotoxicity

3D neural cells

neural cells grown in a 3D environment to mimic the brain and nervous system

hNPC abbreviation

Human neural progenitor cell

cytotoxicity

cell toxicity

LN229 cells

human glioblastoma cell line that are used to study the genetics, cellular dynamics, and treatment response of glioblastoma.

LN229 morphology

epithelial

LN229 origin

Isolated in 1979 from a 60-year-old white female patient with glioblastoma

Stem Cell

cell with the ability to develop into many different cell types in the body. They serve as a sort of internal repair system, capable of dividing and renewing themselves for long periods.

What cells did you put into the 8 chamber chamber slides?

LN229

Where do the particular cells from LN229 come from?

human brain

1Choose macromolecules that considered protein-based and not sugar-based or synthetic:

Laminin

Collagen

Trypsin

Gelatin

Which two protein markers are considered neural stem cells markers

a) Tubb3

b) Cd45

c) Syn

d) Ssea4

e) Sox2

f) Nestin

Sox2 Nestin

Sox2

transcription factor associated with maintaining pluripotency and self-renewal in stem cells

Nestin

intermediate filament protein that is expressed in neural stem cells and neuroectodermal cells during development

Choose macromolecules that considered not protein-based and sugar-based or synthetic:

a.Laminin

b. Collagen

c. Trypsin

d. Heparin

e. Hyaluronan

f. Polypropylene

g. Gelatin

h. Poly(ethylene glycol)

Polypropylene

Poly(ethylene glycol)

Which proteins markers are pluripotency markers? Chooses all the apply.

a) MyyC

b) KLF4

c) Sox2

d) OCT4

e) Map2

f) Sox1

c-myc

KLF4

Sox2

OCT4

Calcein AM

non-fluorescent dye that is hydrolyzed by intracellular esterases in live cells to produce the fluorescent calcein, thereby indicating cell viability

Choose two molecules that would chemically change (reduce/oxidize, hydrolzyed/condensed) in the prescene of living cells

Resazurin
Calcein AM

What two antibiotic reagents are supplemented into RWO medium or 10FD medium to prepare RWOPS or 10FDPS medium

a) Streptomycin

b) BDNF (Brain derived neurotrophic factor)

c) TGF-beta

d) Pencillin

e) Acetaminophen

f) Heparin

g) Clindamycin

h) Amphoteric B

Streptomycin

Pencillin

What are two chemical products that are important to utilize while successfully passaging human pluripotent stem cells (hPSCs) to detach and then adhere to tissue culture plastic (TCP)? Choose from the list below.

a)         Polystyrene

b)         Trypsin-EDTA

c)         ReLeSR

d)         Matrigel or Geltrex

e)         Jell-O

f)          Accutase

Matrigel

Accutase

What media did you use for LN229s

10FD

What is the cell type that you are really actually going to use in the MicroPorject that you and your partner really actually agreed to?

LN229 and RenCell VM

What is the major (one) most important characterization you will perform on the cellular products such as mRNA and proteins?

Gene Expression

What two or three cell culture have you done on Monday (02/17/25)?

LN229 and RenCell VM

If the cells doubled every 24 hours and, if you plated 5,000 cells per well on Monday, what would be the amounts of cells that you predict would be in each well on Wednesday?

20,000

Where do the particular cells from LN229 come from?

Human Brain

What are the cell culture media conditions for your MicroProject?

10FD for LN229, RBE for ReNcell VM

What would happen to a stem cell that gets injected into a human being?

Therapeutic Effects

Immune Response

Tumor Formation

Tissue Integration

No Effect

Ethical and Regulatory Considerations

are LN229 cells stem cells?

No, LN229 cells are not stem cells; they are a human glioblastoma cell line derived from a brain tumor.

What stem cell type are you using for your MicroProject?

ReNcell VM

What is the molecular you use to conduct cell viability experiments? 

Resazurin

What if the chemical compound you are assessing its toxic potential?

LAP (+)

What are the two growth factors in RBE media?

Epidermal Growth Factor (EGF)

Fibroblast Growth Factor (FGF)

Epidermal Growth Factor (EGF):

EGF is a peptide that stimulates cell growth, proliferation, and differentiation by binding to its receptor, EGFR.

Fibroblast Growth Factor (FGF):

family of growth factors involved in various biological processes, including cell growth, development, and tissue repair. FGF2 (also known as basic FGF or bFGF) is commonly used in culture media to support the proliferation and maintenance of stem cells, including neural stem cells.

How to visualize nuclei nuclear stains

Cell samples: This could be cultured cells on coverslips or tissue sections.

Nuclear stain: Common stains include:

DAPI (4',6-Diamidino-2-Phenylindole): Binds to DNA and fluoresces blue.

Protect from light

If Jell-O is a hydrogel, do you think Matrigel is also a hydrogel, after Gelation?

Yes After gelation, Matrigel behaves as a hydrogel since it maintains a water-rich environment and has a 3D structure that can support cell culture. Its unique composition and properties distinguish it from simpler hydrogels like Jell-O

Do you think Matrigel products can be used for 3D cultures because they gel, if the condition is optimal for gelation?

Yes, due to their ability to gel and form a supportive extracellular matrix (ECM) environment.

What are stem cells compared to cancer cells?

Stem cells are undifferentiated cells with the ability to develop into various specialized cell types. They can self-renew (divide to produce more stem cells) and differentiate into specialized cells (like muscle, nerve, or blood cells).

Cancer cells are cells that have undergone mutations leading to uncontrolled growth and division. They may lose normal regulatory mechanisms that control the cell cycle, apoptosis (programmed cell death), and differentiation.

Provide your teams sbel proposal title

Bioprinting & Gene Expression Analysis of ReNCells in 2D vs 3D Cultures

word based abstract

We want to find a significant difference in the gene expression between 2d vs 3d cell culture with ReNcell VM cell lines. The experiment will be performed with the culturing of both cell types into both Matrigel (3D) with Bioprinting and qPCR and Matrigel (2D) into 6 Well Plates. ReNcell VM will be bioprinted in preparation for qPCR analysis. The biomaterials that will be used for bioprinting are GelMA, LAP (+), and Hyaluronic acid. The primers that will used are OCT4, SOX2, NESTIN, TUBB3, and GFAP. ACTB and GAPDH will also be utilized as housekeeping primers. LN229 cells will be used for maintenance and a qPCR control qPCR will be used for gene expression analysis, where the data will be exported into Excel+JMP

What cell dissociation reagent is the most ideal to use with LN-229 cells?

Trypsin

GelMA

modified form of gelatin that has been functionalized with methacryloyl groups.

methacryloyl groups allow for crosslinking when exposed to UV light, forming a hydrogel

GelMA chemical structure

Gelatin Backbone: Gelatin is a protein obtained from collagen, composed of amino acids linked by peptide bonds.

Methacryloyl Groups: These are typically introduced onto the amine groups of the gelatin. The methacryloyl group can be represented as -C(=CH2)CO-.

methacryloyl group

-C(=CH2)CO-

LAP (+) role in bioprinting

photoinitiator to initiate the polymerization of monomers in the presence of light, typically ultraviolet (UV)

LAP (+) chemical structure

Lithium phenyl-2,4,6-trimethylbenzoylphosphinate

hyaluronic acid chemical structure

(C14H21NO11)n

occurring polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine

two protein markers for ReNcell VMs

Nestin and SOX2

LN229 protein markers

EGF

p53

Nestin

GFAP

Pluripotent stem cells (PSCs) protein markers

Oct4

Sox2

Klf4

Klf4

Klf4: A transcription factor involved in maintaining pluripotency

What kind of stem cells are involved in your teams  sbel microproject?

neural stem cells

You can do PCR directly RNAS, true or false.

PCR (Polymerase Chain Reaction) can be performed directly on RNA, but it requires a preliminary step because PCR is designed for DNA amplification. To amplify RNA, you first need to convert it into complementary DNA (cDNA) using reverse transcription (RT). Once you have the cDNA, you can then use standard PCR techniques to amplify the cDNA. This combined process is often referred to as RT-PCR (Reverse Transcription PCR).

What are the 8 chamber well slides for?

Immunocytochemistry

Primary composition of Matrigel

Laminin (60) Major structural and signaling component

Collagen (30) Provides mechanical stability

Entactin (8) Functions as a crosslinker between laminin and collagen IV

Perlecan (2-3%) A heparan sulfate proteoglycan involved in ECM interactions

Laminin

Major structural and signaling component

Entactin

Functions as a crosslinker between laminin and collagen IV

Functions as a crosslinker between laminin and collagen IV

Entactin

Perlecan

A heparan sulfate proteoglycan involved in ECM interactions

A heparan sulfate proteoglycan involved in ECM interactions

Perlecan

gelma crosslinking

important for creating hydrogels, crosslinked using light-activated polymerization. When exposed to UV or visible light, the methacryloyl groups in GelMA undergo free-radical polymerization,

DAPI

4',6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to DNA. It is commonly used in fluorescence microscopy and flow cytometry for labeling nuclear material in cells. DAPI emits blue fluorescence when bound to DNA

PSC vs LN229 vs ReNcell VM

Pluripotent Stem Cells: Broad differentiation potential, used in a variety of research areas including regenerative medicine.

LN229 Cells: Specialized glioblastoma cell line, primarily used in cancer research to study tumor biology and treatment responses.

ReNcell VM: Neural stem cell line, used for studying neural development and diseases, with the ability to differentiate into various neural cell types.

PSC vs LN229 vs ReNcell VM in resazurin assay

PSCs and ReNcell VM may be more sensitive to environmental changes, affecting the reliability of the resazurin assay results. LN229 cells, being a cancer cell line, may provide more consistent results.

mRNA isolation

cells are lysed, and mRNA purified using oligo-dT beads

expression challenges in stem cells

gene expression change due in environmental factors, including repeated passaging

pluripotency genes

oct4

sox2

c-myc

klf4

gapdh

glyceraldehyde-3-phosphate dehydrogenase

ipscs rely on glycolysis for energy important

GFAP

intermediate filament protein expressed in astrocytes in cns

Pluripotent Stem Cells

PSCs are cells that have the ability to differentiate into any cell type of the three germ layers (ectoderm, mesoderm, and endoderm). They can give rise to nearly all cell types in the body.

TUBB3

cytoskeletal protein used a marker for neuronal differentiation

how is uv crosslinking in bioprinting important in sterilization

UV light has germicidal properties that can effectively kill or inactivate bacteria, viruses, and other pathogens

Physical Characteristics of hPSC in Culture

Large nucleus with prominent nucleoli

High nucleus to cytoplasm ratio

Densely packed cells

Defined borders

Was LAP toxic based on your Round 1 cytotox experiment

yes, as it used up a lot of resazurin

What would happen to human stem cells such as ReNcell VM cells and iPSCs that are normally cultured in RBEPS medium or mTeSR Plus Complete medium or Stem Flex Complete medium or StemFlex Complete medium, if key growth factors are excluded? For instance, switching from RBEPS to RWOPS medium and maintaing periodic media change activities with RWOPS. Another example is, for iPSCs, switching from mTeSR Plus medium to neural induction medium or mesenchymal induction medium or cardiomyocyte differentiation kit. Choose all possible answers.

a. Cells will undergo cytofreezing

b. Cells will continue metabolizing

c. Cells will undergo Differentiation

d. Cells will undergo Fixation

e. Cells will undergo Apoptosis

f. Cell will want to Live free

Cells will continue metabolizing

Cells will undergo Differentiation

Cells will undergo Apoptosis

Explain the resazurin assay that your team utilized in the SBEL course to quantitatively analyze cell viability of LC-229 cells.

100 ul of LAP (+) was put as photoinitator for free radicals, then 10 ul of resazurin and 90 ul of media was put in and incubated for 24 hours. the cells acted as an reductase as resazurin catalyzed into resorufin. the sig. difference went down as resazurin ran out

Is your team doing qPCR, what is that for your MicroProject

to find the significant difference between 2d vs 3d cell culture for ReNcell VMs

What are the specific aims for your MicroProjects?

We’re going to perform cell culture + proliferation in order to prepare an aggregate for bioprinting in preparation for qPCR starting mRNA extraction to cDNA synthesis using Nestin, SOX4, OCT4, GAP2, and TUBB3 and housekeeping genes are GAPDH and ACTB.

What is the most expensive reagent for your MicroProject?

ReNcell VM

GFAP (Glial Fibrillary Acidic Protein): expressions in each

PSCs: Typically low or absent expression.

LN229: High expression, as they exhibit characteristics of glial cells.

ReNcell VM: Moderate to high expression in differentiated astrocytes.

SOX2 expressions in each

PSCs: High expression, crucial for maintaining pluripotency.

LN229: Low expression; can vary based on specific conditions or subpopulations.

ReNcell VM: Moderate expression in undifferentiated neural stem cells.

OCT4 expressions in each

PSCs: High expression, essential for pluripotency.

LN229: Generally low expression.

ReNcell VM: Low expression, may vary with differentiation status.