Researchers have cracked the code to doing stem cell transplants and gene therapy without radiation and chemotherapy

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Researchers at Stanford and the University of Tokyo may have cracked the code to doing stem cell transplants and gene therapy without radiation and chemotherapy.

For decades, researchers have been stymied in their attempts to grow large numbers of hematopoietic stem cells in the laboratory.

These rare bone marrow cells are solely responsible for generating all the cells of the blood and immune system.

Difficulties in growing the cells have seriously hampered many research efforts, including those aimed at making stem cell transplantation or gene therapy in patients with certain cancers or blood disorders easier and safer.

Now, researchers at the Stanford University School of Medicine and the University of Tokyo have cracked the code.

By tinkering with the components of the nutritive broth in which the cells are grown, the specialized molecules used to support their growth and the physical conditions under which the cells are cultivated, the researchers have shown for the first time that it’s possible to coax hematopoietic stem cells from mice to renew themselves hundreds or even thousands of times within a period of just 28 days.

a, Schematic of the standard HSC culture assay. Bone-marrow CD34−KSL HSCs of C57BL/6-CD45.1 mice were sorted (50 cells per well) into U-bottomed 96-well plate wells (b for sorting scheme). HSC growth can be observed during culture by counting or flow cytometry, with medium changes made every three days (after an initial seven days in culture). After 7–28 days, functional HSC activity was determined using competitive transplantation into irradiated C57BL/6-CD45.2 mice, against 1 × 106 bone-marrow competitor cells from C57BL/6-CD45.1/CD45.2 (F1) mice. Donor chimerism within peripheral-blood myeloid, T cell and B cell lineages was determined after 4–16 weeks, or longer. Where indicated, secondary transplantation assays were performed by transplanting 1 × 106 bone-marrow cells from primary recipients into irradiated C57BL/6-CD45.2 mice. b, FACS gating strategy for sorting CD34−KSL cells (gates 1–7) and CD150+CD34−KSL cells (gates 1–8) from c-KIT-enriched mouse bone marrow. Representative of at least five experiments. c, Flow cytometric histograms for cell-surface c-KIT staining of HSCs following stimulating with 100 ng ml−1 TPO and 0, 10 or 100 ng ml−1 SCF for 1, 24 and 48 h. Representative of three independent cultures. d, Mean florescence intensity of c-KIT antibody staining on HSCs cultured in 100 ng ml−1 TPO supplemented with 10 ng ml−1 or 100 ng ml−1 SCF, analysed after 1–72 h in culture, relative to cultures containing 100 ng ml−1 TPO without SCF. Mean of three independent cultures. Error bars denote s.d. e, Mean donor peripheral-blood chimerism at week 16, from 1 × 104 HSC-derived cells following a 28-day-long culture on plastic (n = 5 cell cultures), collagen 1 (n = 3 cell cultures), collagen 4 (n = 4 cell cultures), fibronectin (n = 3 cell cultures), gelatin (n = 5 cell cultures) or laminin 511 (n = 4 cell cultures) culture plates (cultured in 100 ng ml−1 TPO and 10 ng ml−1 SCF with complete medium changes). Competitive transplantation against 1 × 106 bone-marrow competitors. f, Number of live cells after culturing 50 CD34−KSL HSCs for 28 days on plastic (tissue-culture-treated) plates or fibronectin-coated plates. Mean of three independent cultures. Error bars denote s.d.

“This has been one of my life goals as a stem cell researcher,” said Hiromitsu Nakauchi, MD, Ph.D., professor of genetics at Stanford.

“For 50 years, researchers from laboratories around the world have been seeking ways to grow these cells to large numbers.

Now we’ve identified a set of conditions that allows these cells to expand in number as much as 900-fold in just one month.

We believe this approach could transform how hematopoietic stem cell transplants and gene therapy are performed in humans.”

In particular, the researchers have shown it is possible to successfully transplant large numbers of the cells into mice without first eliminating the recipients’ own stem cell population.

If the technique also works in humans, it could save thousands of patients with blood or immune disorders from a grueling regimen of radiation or chemotherapy prior to transplant.

It could also allow clinicians to use a patient’s own genetically corrected stem cells for gene therapy.

The study was published online May 29 in Nature. Nakauchi shares senior authorship of the study with Satoshi Yamazaki, Ph.D., an associate professor of stem cell biology at the University of Tokyo.

Postdoctoral scholar Adam Wilkinson, Ph.D., of Stanford and senior research assistant Reiko Ishida of the University of Tokyo are the lead authors.

Hematopoietic stem cells are rare cells found in the bone marrow.

Like other stem cells, they can either divide to make more hematopoietic stem cells – a process called self-renewal – or generate the precursors of all the different types of blood and immune cells in the body – a process called differentiation.

It’s long been known that people with immune or blood disorders such as sickle cell anemia or leukemia can be cured with a transplant of healthy hematopoietic stem cells.


Hematopoietic stem cell (HSC) research took hold in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production.

Attempts to quantify the cells responsible led to the discovery of serially transplantable, donor-derived, macroscopic, multilineage colonies detectable on the spleen surface 1 to 2 weeks posttransplant.

The concept of self-renewing multipotent HSCs was born, but accompanied by perplexing evidence of great variability in the outcomes of HSC self-renewal divisions.

The next 60 years saw an explosion in the development and use of more refined tools for assessing the behavior of prospectively purified subsets of hematopoietic cells with blood cell–producing capacity.

These developments have led to the formulation of increasingly complex hierarchical models of hematopoiesis and a growing list of intrinsic and extrinsic elements that regulate HSC cycling status, viability, self-renewal, and lineage outputs.

More recent examination of these properties in individual, highly purified HSCs and analyses of their perpetuation in clonally generated progeny HSCs have now provided definitive evidence of linearly transmitted heterogeneity in HSC states.

These results anticipate the need and use of emerging new technologies to establish models that will accommodate such pluralistic features of HSCs and their control mechanisms.

Human Cells

The gold standard assay to experimentally test the in vivo repopulating potential of human HSCs is intravenous or intra-bone injection into sublethally irradiated, genetically immune-deficient mice.

Successful engraftment of human stem cells is defined by the detection of a threshold number of human blood cells (typically >0.1% of nucleated cells) in the blood, BM or other mouse organs several weeks to months after transplantation using flow cytometry.

As with mouse transplantation assays described above, xenotransplantation assays can be performed under limiting-dilution conditions to determine the frequency of repopulating stem cells in human hematopoietic tissues and purified cell populations.

Originally most xenotransplantation assays were performed in the SCID and NOD/SCID mouse strains. In these older studies relatively large numbers of cells were required to overcome immune rejection by residual host macrophages and NK cells.

Moreover, human hematopoiesis could only be detected during a relatively short period (6 – 12 weeks) due to the short lifespan of the mice.

Due to these limitations it was not possible to study the kinetics of human cell engraftment or to distinguish between HSC subsets that mediate short-term and long-term reconstitution.

Some of these difficulties of xenotransplantation assays have been addressed by the development of mouse strains in which more immunomodulatory cell types have been deleted.

The new immunodeficient mouse strains also live longer than the original strains.

Specifically, b2-microglobulin-deficient and interleukin (IL)-2 receptor (R) γ-deficient NOD/SCID mice support high levels of engraftment that can be detected for >20 weeks after transplantation.7-10 IL-2Rγ deficient mice with functional impairment of endogenous HSCs due to loss-of-function mutations in the c-kit gene are even more permissive for human HSC engraftment and do not require pre-transplant conditioning by irradiation.11

Using these newer mouse strains it is now possible to study the properties of human HSCs in great detail and identify HSC subsets with distinct cell surface marker profiles, lineage potentials and engraftment kinetics.12-16


But in order for the treatment to work, the recipient’s own hematopoietic stem cells must be killed to eliminate the disease and make space for the healthy cells to settle in the bone marrow.

This elimination step, also called “conditioning,” is accomplished with either chemotherapy or radiation, or a combination of the two. The conditioning, however, can cause life-threatening side effects.

This is particularly true in pediatric patients, who can suffer side effects such as growth retardation, infertility and secondary cancer in later life. Very sick or elderly patients often can’t receive transplants because they are unable to tolerate the conditioning treatment.

Radiation-free stem cell transplants, gene therapy may be within reach
a, Fold change in MFI from cytokine immunoassays performed on HSA-based HSC cultures between day 8 and day 13. Medium changes performed at day 7 and day 10. Mean of four independent cultures with fold change relative to unconditioned medium. Error bars denote s.d. b, Mean expansion of 50 CD34+KSL haematopoietic progenitor cells at day 7, in 100 ng ml−1 TPO and 10 ng ml−1 SCF with or without addition of 0.3 ng ml−1 to 10 ng ml−1 mouse IL-6 (n = 4 cell cultures). c, Heat map displaying the MFI fold change from cytokine immunoassays using conditioned medium from HSC cultures at day 14. CD34−KSL HSCs were isolated from C57BL/6 wild-type (WT), Tlr2 knockout (TLR2-KO) or Tlr4 knockout (TLR4-KO) mice, and cultured in HSA-based cultures. Dexamethasone (+Dex) at 50 nM was added, where indicated. Mean of four independent cultures with fold change relative to unconditioned medium. d, Concentration of IL-6 observed in HSA-based cultures at day 14 of wild-type HSCs (n = 8 cell cultures), Tlr2 knockout HSCs (n = 8 cell cultures), Tlr4 knockout HSCs (n = 6 cell cultures), or wild-type HSCs + dexamethasone (n = 8 cell cultures). Error bars denote s.d. e, Mean donor peripheral-blood chimerism at week 12, from HSCs cultured for 7 days, in fresh medium (n = 7 mice) or in medium composed of 50% medium collected from a 12-day-long HSC culture and 50% fresh medium (termed ‘conditioned media’, n = 7 mice). Competitive transplantation against 1 × 106 bone-marrow competitors. f, Example flow cytometry plots displaying c-KIT and SCA1 expression on the Lin− progeny (left), and CD150 and CD48 expression in the KSL population (right) after a PVA-based HSC culture for seven days. Representative of four independent cultures. g, Concentration of various cytokines in conditioned medium at day 14, from HSA- or PVA-based CD34−KSL HSC cultures. Mean of eight independent cultures. Error bars denote s.d. Statistical significance was calculated using t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. h, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (HSA-based cultures with half-medium changes, HSA-based cultures with complete medium changes and PVA-based cultures with complete medium changes), relative to expression in freshly isolated KSL cells. Mean of three independent cultures, with gene expression normalized to Gapdh expression. Error bars denote s.d. i, Number of phospho-γ-histone 2A.X (H2A.X) nuclear foci in KSL cells at day 28, from HSA-based or PVA-based HSC cultures. Irradiated cells were included as a positive control. Forty-nine cells quantified per condition. j, Relative expression of p16Ink4a, p19Arf and Trp53 in KSL cells collected from cultures at day 14 (left): HSA-based cultures, PVA-based cultures and PVA-based cultures supplemented with 1 ng ml−1 lipopolysaccharide. Mean of technical quadruplets, with gene expression normalized to Gapdh expression. The concentration of IL-6 observed in these culture conditions is shown on the right. Mean of four independent cultures. Error bars denote s.d. k, Twenty-eight-day-long expansion of 50 CD150+CD34−KSL HSCs in medium containing 87% hydrolysed PVA or >99% hydrolysed PVA. Ten thousand cells at day 28 represent ~1 HSCeq for 87% PVA and ~5 HSCeq for 99% PVA. Mean of three independent cultures. Error bars denote s.d. l, Seven-day-long expansion of 50 human cord-blood CD34+ cells in HSA- or PVA-based cultures supplemented with 10 ng ml−1 human SCF and 100 ng ml−1 human TPO. Mean of three independent cultures. Error bars denote s.d.

“A Holy Grail in the stem cell field’

But for some time, researchers have wondered whether transplanting large numbers of donor hematopoietic stem cells could circumvent the need to remove the existing cells.

Perhaps, they reasoned, swamping the recipient’s bone marrow with a tide of healthy donor cells would allow the newcomers to muscle their way in and set up shop making healthy blood and immune cells.

It’s been difficult to test this theory, however, because hematopoietic stem cells are hard to isolate in large numbers.

And although it’s been possible for years to grow human hematopoietic stem cells in the laboratory, the cells never self-renew robustly and instead often abandon their stem cell fate to differentiate into precursor cells.

As a result, it’s been difficult to study their biology in depth or to generate enough cells to attempt large-scale transplants.

“Expansion of hematopoietic stem cells has been a Holy Grail in the stem cell field,” Nakauchi said.

“When something does not work, people tend to think that something is missing.

But we decided to try the opposite approach by eliminating all the impurities in the conventional culture system, while also optimizing other aspects that encourage self-renewal of the hematopoietic stem cells.”

The researchers also replaced a potentially contaminated blood protein called serum albumin with polyvinyl alcohol, a water-soluble synthetic chemical that is frequently used in biomedical research.

These modifications to how the cells were grown allowed the researchers to generate enough hematopoietic stem cells for transplant from just 50 starting cells.

Although some of the cells did differentiate in culture, many more than normal maintained their stem cell identity throughout the duration of the culture.

“These original 50 cells increased in number about 8,000-fold over 28 days,” Nakauchi said. “Of these, about one of every 35 cells remained a functional hematopoietic stem cell.”

Planning to test human cells

The researchers estimated that the hematopoietic stem cells in the original sample increased in numbers by between 200- to 900-fold – an unprecedented level of expansion.

When they transplanted the newly grown cells into mice that had not undergone a conditioning regimen, the animals developed blood and immune cells derived from both the donors’ hematopoietic stem cells and their own, demonstrating that the donor cells had engrafted and remained functional.

“We also found that, during the culture, we can use CRISPR technology to correct any genetic defects in the original hematopoietic cells,” Nakauchi said.

“These gene-corrected cells can then be expanded for transplantation. This should allow us to use a patient’s own cells as gene therapy.”

Nakauchi and his collaborators at Stanford are now testing this approach in mice.

More information: Adam C. Wilkinson et al. Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation, Nature (2019). DOI: 10.1038/s41586-019-1244-x

Journal information: Nature
Provided by Stanford University Medical Center

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