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Cell Biology > Stem cells and cell differentiation > Flashcards

Stem cells and cell differentiation Flashcards

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1
Q

Dr. Alfred Cioffi

A

A priest who got his PhD in molecular biology at age 54

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2
Q

Uses for stem cells (8)

A
  1. Increased understanding of how diseases develop
  2. Cure diseases
  3. Test new drugs for safety
  4. Generate new stem cells to replace or aid diseased or damaged Cells
  5. Research how certain cells develop into cancer
  6. Regenerative medicine applications
  7. Fix genetic diseases
  8. Clean meat industry
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3
Q

Printing of human organs in space

A

Biological printing of the tiny, complex structures found inside human organs, such as capillaries, has proven difficult in Earth’s gravity. Under Earth’s gravity, an initial scaffolding (support structure) is necessary to form the desired shape of the tissue. The BioFabrication Facility (BFF) attempts to take the first steps toward printing human organs and tissues in microgravity using
ultra-fine layers of bioink that may be several times smaller than the width of a human hair. This research is part of a long-term plan to manufacture entire human organs in space using refined biological 3D printing techniques

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4
Q

Engineered heart tissues

A

This study looks at how human heart tissue functions in space. It uses unique 3D tissues made from heart cells derived from human induced Pluripotent Stem Cells (hiPSCs), essentially adult stem cells. The engineered heart tissues, or EHTs, are complex 3D structures, each about the size of a few grains of rice. These structures are more similar to tissues in the body than flat cell cultures in a petri dish or those floating in a flask of liquid

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5
Q

Why can mesenchymal stem cells be used to treat covid?

A

MSCs are well-known for their immunomodulatory and regenerative potential. In a recent Phase I/II clinical trial for Rheumatoid Arthritis, results appeared to show that HB-adMSCs were safe and effective in attenuating systemic
inflammation. In COVID-19 patients, inflammation is a
driving force behind disease progression, and it is critical to regulate the immune system as early as possible.

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6
Q

Hope biosciences

A

Hope Biosciences, a clinical stage biotechnology company
focused on developing cell-based therapeutics for acute and chronic disease, announced that FDA has approved a Phase II clinical trial evaluating efficacy and safety of Hope Biosciences’ autologous, adipose-derived mesenchymal stem cells (HB-adMSCs) to provide immune support against COVID-19. It is hoped that this pretreatment will limit the progression and severity of COVID-19, ultimately keeping patients out of the hospital and off of mechanical ventilation.

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7
Q

Stem cell

A

A cell that can renew (divide) or differentiate- these cells can take 2 different pathways to either specialize or remain stem cells. The number of doublings is influenced by source and type. hESC and iPSCs are immortal, adult sourced have 50-100-200 doublings (approximate)

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8
Q

4 categories of stem cells

A
  1. Adult stem cells
  2. Fetal stem cells
  3. Embryonic stem cells
  4. Induced pluripotent stem cells
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9
Q

Adult stem cells

A

Most popular are adipose (fat) derived mesenchymal stem cells (adMSCs) now in more than 700 stem cell therapy trials globally. These stem cells are most commonly used for stem cell therapy and can be autologous

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10
Q

Fetal stem cells

A

Can be amniotic, from the umbilical cord, or from the placenta

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11
Q

Embryonic stem cells

A

Includes hESCs and hPSCs, with hESCs in clinical trials as of 2010

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12
Q

Induced Pluripotent Stem Cells

A

Not in clinical trials in US but patients being treated in Japan and Australia. Adult cells are exposed to IPS reprogramming factors to convert them to IPS cells, which act like stem cells and differentiate.

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13
Q

Differentiation

A

When a cell becomes more specialized, such as a fibroblast or a hepatocyte. However, differentiation can be partial or full. We have to use molecular metrics to compare one iPSC generated hepatocyte to another iPSC generated hepatocyte. Some stem cells are progenitor cells and can only make one or two types of cells, while others are totipotent

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14
Q

Transdifferentiation (Direct Reprogramming)

A

Ability of a differentiated cell to become another type of differentiated cell without going through an
embryonic step (e.g. unlike iPSCs). This can be induced in vitro but not sure if this happens in vivo. First done experimentally in 1987 but several cells have been generated since that time

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15
Q

Dedifferentiation and Redifferentiation

A

Ability of a cell to become more embryonic-like and differentiate into another cell type. Chemicals like “reversine” can induce de-
differentiation

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16
Q

Which animals can regenerate lost limbs?

A

Red spotted newts can regenerate lost limbs and lenses of the eye

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17
Q

Stem cell niche

A

Also called the stem cell microenvironment, critical for controlling cell division vs differentiation. The stem cell niche is very complex, and includes neighboring cells, ECM, local growth factors (FGF, others), physical environment (pH, oxygen tension, pressure)

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18
Q

Totipotent

A

Stem cells that can differentiate into any cell type. This is the highest level of “stemness”.

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19
Q

Pluripotent

A

Stem cells can differentiate into many cell types. Restricted stemness

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20
Q

Multipotent

A

Stem cells can differentiate into several cell types. Stemness is even more restricted

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21
Q

Unipotent

A

Stem cells can only differentiate into one type of stem cell.

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22
Q

Blastocyst

A

Late pre-implantation stage embryo. hESCs originate from inner cell mass

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23
Q

Chimera test

A

Only true test of totipotency of a candidate stem cell. Implant test stem cell in blastocyst and then implant chimeric
embryo in surrogate mother. Then the cell is tracked in all tissues and organs of newborn using GFP. This is not legal in humans and has only been done in mice. Therefore, we can say that mESC (mouse) are totipotent, but can’t be sure of human ESC.

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24
Q

Biodistribution

A

Ability of stem cells to find “home” – its targeted tissue. Damaged or compromised tissue releases factors that causes endogenous MSCs to home to damaged site. Occurs in vivo- transplanted XX hearts in XY patients have XY cardiomyocytes upon autopsy (10%) – a clear demonstration of endogenous stem cell homing and repair

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25
Q

Shinya Yamanaka

A

Given the Nobel prize in 2012 for his research on induced pluripotent stem (IPS) cells. iPS reprogramming factors are introduced to adult cells, creating IPS cells. IPS cells are embryonic stem cell-like cells that can differentiate into other types of cells.

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26
Q

STAP cells

A

Stimulus triggered acquisition of pluripotency cells. In this study, cells were isolated and acid was added to shrink the cells and “shock” them to revert them to a stem cell-like state. These findings were reported in 2014 but were later retracted as the research was fraudulent.

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27
Q

Fusogenic

A

This is problem with stem cells- they can spontaneously fuse with each other, forming a tetraploid cell (could generate cancer stem cells). When injected into patients mechanical stress can cause fusion

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28
Q

Bioethics

A

The norms of conduct. Relatively term and country dependent

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29
Q

Therapeutic cloning

A

The production of embryonic stem cells for the use in replacing or repairing damaged tissue or organs. The embryo is developed under laboratory conditions and treats diseases like diabetes and Alzheimer’s Disease. An egg’s nucleus is removed and a diploid nucleus from a body cell is transferred into the egg to create an embryo that stem cells can be harvested from.

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30
Q

Reproductive cloning

A

The deliberate production of genetically identical individuals, each newly produced individual is a clone of the individual. The embryo that is created is developed under uterine conditions. A diploid nucleus is still inserted into an enucleated egg, but the egg is implanted into a surrogate mother (like a sheep), and the clone will be born. This method is used to harvest stem cells that can be used to study embryonic development.

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31
Q

SCID mice

A

Severe combined immunodeficiency- have no B and T cells and thus have a compromised immune system. Are used for determining if an injected candidate stem cell can differentiate
in vivo into a multitude of tissue types in vivo. Are also used to determine if a candidate human cancer cell can generate tumors in vivo

32
Q

3 ways to generate stem cells in the laboratory

A
  1. Somatic Cell Nuclear Transfer (SCNT)
  2. Parthenogenesis (hPSCs)
  3. Induced Pluripotent Stem Cells (iPSCs)
33
Q

Somatic cell nuclear transfer

A

A technique in which the nucleus (DNA) of a somatic cell is transferred into an enucleated metaphase-II oocyte for the generation of a new individual, genetically identical to the somatic cell donor. This has been used for reproductive cloning, like when Dolly the sheep was cloned

34
Q

Which animals have been closed using SCNT?

A

Sir Ian Wilmut cloned Dolly in 1996 but John Gurdon did it with frogs in 1960 and finally was awarded a Nobel Prize in 2012. First pet clone was a cat- “Little Nicky” – 2004

35
Q

Challenges of SCNT

A

1000s SCNT are required for one implantable embryo. This is still the problem today- as a result many SCNT researchers left the field. But some are still attempting human SCNT designed
for therapeutic cloning because hESCs could serve as an autograft

36
Q

SCNT for autologous therapeutic cloning

A

Take out a somatic cell (the person’s own cell). Use SCNT and introduce the cells back into the person.

37
Q

Dolly the sheep

A

Dolly was born at Edinburgh University in 1996, she was euthanized at age 6 due to lung disease and advanced arthritis. But other identical clones, now 9 years old, from the same
SCNT (Debbie, Denise, Diana and Daisy) are fine. SCNT showed that a SCNT nucleus could create an entire functioning animal due to cytoplasmic factors in the egg

38
Q

Cloning of primates

A

Monkeys were cloned in 2018 using SCNT

39
Q

Parthenogenesis

A

A type of asexual reproduction- development of an embryo from an unfertilized egg cell (no sperm). May be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells.

40
Q

Experimental parthenogenesis

A

Artificial parthenogenesis was first shown in 1913 by Loeb using two experimental systems. Unfertilized sea urchin eggs were induced to undergo parthenogenesis by changing the osmolarity of the surrounding medium. Unfertilized starfish eggs could do the same using dilute acid

41
Q

Human parthenogenetic stem cells (hPSCs)

A

Also called Parthenotes. Derived from the inner cell mass of blastocysts obtained from unfertilized oocytes that have been activated using parthenogenesis. These cells demonstrate characteristics typical for human embryonic stem cells (hESC), including extensive self-renewal and in vivo as well as in vitro differentiation into cells of all three germ layers.

42
Q

Benefit of hPSCs

A

According to ISCO- only 200 to 300 eggs would be required to generate hPSCs that could match anyone in the world

43
Q

Limitations of hPSCs (3)

A
  1. All alleles will be homozygous because of no sperm
  2. Not FDA approved in US
  3. Is it ethical to create a human embryo?
44
Q

Autologous stem cell transplant process using induced pluripotent stem cells

A

Patient cells are harvested and made into induced pluripotent stem cells. A genetic mutation can be targeted and repaired. Then, the now healthy stem cells undergo in vitro differentiation. Healthy, genetically matched cells are transplanted back into the patient.

45
Q

How can induced pluripotent stem cells be used to test drugs?

A

Once patient IPS cells are generated, they will undergo in vitro differentiation. The affected cells are then screened for therapeutic compounds. The patient can be treated with whatever compound is found to be effective.

46
Q

Converting human somatic cells to stem cells using 4 genes

A

Two groups have shown that somatic human cells can be converted to true stem cells (iPSCs) with only 4 additional genes. Shinya Yamanaka of Kyoto University in Japan
used a retrovirus to ferry into adult human
fibroblasts OCT3/4, SOX2, KLF4 and c-MYC
genes. Sources were skin cells from 36 year old female and 69 year old man. Also, James Thomson of the University of Wisconsin (pioneer in hESCs) used OCT3/4, SOX2, NANOG and LIN28 genes

47
Q

How do genes influence the development of iPSCs?

A

There are activate genes that code for self renewal and pluripotency. There are also repress genes that induce specific differentiation pathways. This information comes from research on SCNT

48
Q

RT-PCR

A

Reverse transcription PCR. Studies true differentiation in culture as revealed by appropriate markers

49
Q

SCID mouse test

A

Is a teratoma
generated by iPSC injection? The iPS cells form teratomas in mice – a non malignant
mass of differentiated cells derived from iPSCs

50
Q

Teratocarcinoma

A

A malignant teratoma that
originates from embryonic cells or stem cells. Can iPSCs and hESCs do this too? In the US, iPSCs are not in clinical trials like human embryonic stem cells

51
Q

Cellular dynamics company

A

Sells iPSCs

52
Q

Uses of iPSCs (4)

A
  1. Basic research on differentiation
  2. Can make patient specific cells of individuals carrying genetic defects
  3. Source of cells in the future for stem cell
    therapy. Not yet FDA approved but Cynata Therapeutics
    (Australia) just completed first clinical trials in using iPSCs.
  4. Will be useful in tissue engineering
53
Q

Maturation Phase Transient Reprogramming

A

Scientists at the Babraham Institute have developed a method to “time jump”
human skin cells (fibroblasts) by 30 years turning back the aging clock. They measured telomere attrition, genetic instability, epigenetic and transcriptional alterations and the accumulation of
misfolded proteins – all accepted markers of cell aging. They used the
same 4 Yamanaka reprogramming factors
but rather than waiting the required 50 days culture time to generate iPSCs they waited just 13 days and the cell has now
reversed its aging process by 30 years

54
Q

Cellular reprogramming and liver regeneration

A

Researchers at Salk lab
used a short-term (one day, not 50) Yamanaka factor protocol to partially
reprogram mice liver tissue. Liver exhibited improved regeneration and younger characteristics. But, they
didn’t generate teratomas
or other cancers which is
typical of a standard longer Yamanaka procedure. Currently trying to understand the molecular basis of this “rejuvenation”

55
Q

SCNT pros and cons (3)

A
  1. Could be used for an autologous transplant if FDA approved
  2. No US federal laws banning therapeutic or reproductive cloning
    but some states forbid it.
  3. But is it ethical? A “human embryo” is being created.
56
Q

Parthenogenesis pros and cons (4)

A
  1. Can match to a world population – only 300 eggs required
  2. But all alleles are homozygous, not heterozygous
  3. Allogeneic, not autologous like SCNT unless female donated egg
  4. But is it ethical? A “human embryo” is being created.
57
Q

iPSCs pros and cons (4)

A
  1. No “human embryo” created as in previous two
  2. Can be autologous or allogeneic
  3. But potential for teratocarcinomas
  4. More pluripotent than fat (adipose)-derived adult mesenchymal
    stem cells and easier to procure
58
Q

Stem cells safety issues (3)

A
  1. Tumorigenicity
  2. Immunogenicity
  3. Inappropriate differentiation
59
Q

Tumorigenicity

A

Stem cells have long telomeres and can divide many more times than normal cells. (Telomeres = “mitotic clock”). Propensity to form tumors and teratomas. One clinical trial started in Japan overseen by the RIKEN Institute (later) was stopped after only one patient due to this concern

60
Q

Immunogenicity

A

Propensity to trigger immune response. The more frequent the stem cell injections the higher the chance of immune rejection complications that could include anaphylaxis. Autologous as well as allogeneic uses can launch an immune response

61
Q

Inappropriate differentiation

A

Risk of stem cells differentiating into cells that were not intended
and not native to target organ. Example- A woman injected with human mesenchymal stem cells
(MSCs) near her eyes ended up with bone tissue growing inside her eyelids

62
Q

Use of stem cells to repair brain damage

A

Transplanting neuronal
stem cells into damaged or diseased areas of the
brain has been touted as a potential therapeutic option, although it has been met with some difficulties. Yet now, investigators at Lund
University in Sweden may have just found a path
forward for using induced pluripotent stem (iPS)
cell-derived cortical neurons for treating stroke-afflicted rats. The approach was able to restore mobility and sensation of touch by reprogramming human skin cells to become nerve cells, which were then transplanted into the rats’ brains

63
Q

3D printing cardiac patches and heart

A

World’s first vascularized human heart with atria and ventricles and patches – heart size of a rabbit heart. It was generated from iPSCs and donor’s own ECM that is processed into a personalized hydrogel making them biocompatible for implantation in the future. Goal is to train them to behave like a real heart.
Cardiomyocytes beat but not in a coordinated fashion

64
Q

Hematopoiesis

A

An example of a unipotent stem cell. BMT- replacing stem cells to make new blood cells, used to treat blood cancers

65
Q

Zebrabow

A

Specially bred (transgenic) zebrafish whose hematopoietic stem cells can fluoresce up to 80
different colors so that stem cell fate can be tracked. Multiple copies of genes for red to blue fluorescent proteins are a permanent part of the genome of Zebrabow. Technique is referred to as “fluorescent bar coding”

66
Q

Cord blood

A

Cord blood contains hematopoietic stem cells, and cord tissue contains mesenchymal stem cells. It can be stored in case the patient is ever in need of stem cells

67
Q

Private cord blood banks

A

Incorporated as a “for profit” organization-donors pay an initial fee and a maintenance fee. Cells not available to the public. Better if there is a genetic disease in the family and multiple members require the cells

68
Q

Public cord blood banks

A

Incorporated as a “not for profit” organization. Available to the public through the National
Marrow Donor Program through which cord
blood is matched

69
Q

ViaCord

A

A private cord blood bank

70
Q

C. elegans

A

Used to study cell differentiation by Robert Korbis. Translucent- made microscopy easier

71
Q

Benefits of C. elegans

A
  1. Easy to grow on agar plates
  2. Non pathogenic
  3. Translucent – can optically section through organism
  4. Stable mutant C. elegans are available for study
  5. All cells have been coded and differentiation predicted
  6. Cell division or differentiation patterns can be predicted and always follow the same pattern
  7. Many genes like the apoptotic genes have mammalian
    homologs. The apoptotic genes were first identified in C. elegans – excellent model to study apoptosis
  8. Comprised of a limited number of cells (about 1000)
  9. First microRNA (miRNA) discovered
72
Q

Cell polarity

A

The ability of cells to reorganize their internal structure, resulting in changes in cell shape and the generation of regions of the plasma membrane with different morphologies and protein and lipid compositions. C.elegans has been used to study the polarization of embryonic axes

73
Q

Par proteins

A

Before its first cell division, the C. elegans zygote is asymmetric, and its first division is asymmetric. Cytoplasmic complexes called P granules concentrate in the region of the cell that gives rise to the posterior end of the embryo. To study this, genetic mutations were identified where there was a symmetric first division of a zygote. The P granules were not positioned correctly, so these genes were called partition defective (Par genes). It was found that par proteins are asymmetrically localized by antagonistic interactions.

74
Q

miRNA and C. elegans

A

Researchers can silence a C. elegans gene of interest using one of two methods (double stranded RNA in vitro or in vivo). The C. elegans heterochronic mutant was key to discovering lin-4 RNA- this was the first microRNA ever discovered

75
Q

MicroRNA

A

Small endogenous RNA molecules that regulate genes

76
Q

Xenobots

A

Scientists at the University of Vermont, Tufts University and Harvard University’s Wyss Institute for Biologically Inspired Engineering said they have discovered an entirely new
form of biological reproduction different from any animal or plant known to science. Frogs have a way of reproducing that they normally use but when you liberate the stem cells from the rest of the embryo and you give them a chance to figure out how to organize
in a new environment, not only do they figure out a new way to move, but they also figure out apparently a new way to reproduce by pushing cells together in a clump to make another C-shaped Xenobot

Cell Biology (12 decks)

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