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Q: Error using the "prob" package in an R function I'm attempting to write a function that uses the prob package to compute conditional probabilities. When using the function I continue to encounter the same error, which states an object within the function cannot be found.
Below is a reproducible example in which I compute a conditional probability without the function and then attempt to use the function to produce the same result. I'm not sure if the error is due to limitations with the prob package or an error on my part.
# Load prob package
library(prob)

# Set seed for reproducibility
set.seed(30)

# Sample data frame
sampledata <- data.frame(
  X <- sample(1:10),
  Y <- sample(c(-1, 0, 1), 10, replace=TRUE))

# Set probability space
S <- probspace(sampledata)

# Subset Y between -1 and 0
A <- subset(S, Y>=-1 & Y<=0)

# Subset X greater than 6
B <- subset(S, X>6)

# Compute conditional probability
P <- prob(A, given=B)

The above code produces the following probability:
> P
[1] 0.25

Attempting to write a function to calculate the same probability:
# Create function with data frame, variables, and conditional inputs
prob.function <- function(df, variable1, variable2, state1, state2, cond1){
  s <- probspace(df)
  a <- subset(s, variable1>=state1 & variable1<=state2)
  b <- subset(s, variable2>cond1)
  p <- prob(a, given=b)
  return(p)
}

# Demonstrate the function
test <- prob.function(sampledata, Y, X, -1, 0, 6)

This function gives the following error:
Error in eval(expr, envir, enclos) : object 'b' not found

Any help you can provide would be great.
Thanks!

A: This looks like a bug in prob.
When I run this in Vanilla R, I get the same error.  But when I create an object b in my workspace, the error disapears:
> print(b)
Error in print(b) : object 'b' not found
> test <- prob.function(sampledata, Y, X, -1, 0, 6)
Error in eval(expr, envir, enclos) : object 'b' not found
>
> b <- "dummy variable"
> print(b)
[1] "dummy variable"
> test <- prob.function(sampledata, Y, X, -1, 0, 6)
> test
[1] 0.25
>

As a temporary workaround, just create a dummy b in your current environment.

As for the bug, if you look at the source for prob.default (which in the example above is what prob(a, given=b) is eventually calling), you'll see the following section:
if (missing(given)) {
    < cropped >
}
else {
    f <- substitute(given)
    g <- eval(f, x)                  <~~~~
    if (!is.logical(g)) {            <~~~~
        if (!is.data.frame(given))   <~~~~
            stop("'given' must be data.frame or evaluate to logical")
        B <- given

    }
    ...
    < cropped >
}

it is jumping from g to given, perhaps inadvertently?    I would reach out to the package maintainer, as this may be an oversight.

A: I don't think this is a bug in package prob.
First, you should create you sampledata as
sampledata <- data.frame(
  X = sample(1:10),
  Y = sample(c(-1, 0, 1), 10, replace=TRUE))

Your original code creates not only this dataframe but also  variables X and Y in the global environment which are actually being used later when you call your function.
Second, you shouldn't call subset() inside a function. Use bracket subsetting instead:
prob.function <- function(df, variable1, variable2, state1, state2, cond1){
  s <- probspace(df)
  a <- s[s[[variable1]]>=state1 & s[[variable1]]<=state2, ]
  b <- s[s[[variable2]]>cond1, ]
  p <- prob(a, given=b)
  return(p)
}

And pass variable1 and variable2 as strings:
test <- prob.function(sampledata, "Y", "X", -1, 0, 6)

Now you have test==0.25, and no error.
References for what is going on:

*

*http://adv-r.had.co.nz/Computing-on-the-language.html#non-standard-evaluation-in-subset

*Assignment operators in R: '=' and '<-'

*Why is `[` better than `subset`?


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There are three methods of handling Employee Receivables, entirely through Payroll, through Payroll and Accounts Receivable and through Financials. Here, we'll be taking a look at the payroll only method.
Using the payroll method, all money is tracked through payroll and a payroll report is available by employee in a consolidated format.
Here's the accounting flow in this scenario: When the loan is entered, Employee Receivables is debited and, in this case, Supplies and Tools will be credited. Employee Receivables will be credited the amount of the deduction from the employee's paycheck.
First, you'll need to set up General Ledger Employee Receivable account in your chart of accounts, located under Company → Enter Chart of Accounts.
Next, you'll set up a Deduction by going to Employees → Additions and Deductions. Set up a deduction called "Employee Receivable" that will be used for the payroll deduction.
Reporting Type should be None.
No items should be checked in Taxes That Apply To This Item.
You'll also need to set up an Addition under Employees → Additions and Deductions. Set up an addition called "Employee Loan" that will be used to enter the amount of the loan you made to your employee.
Company Paid should be checked and the account in this field should be the account that was debited when the loan was made (in this example Tools & Supplies).
Entering or recording the loan can be done a couple of different ways. One way is from the timesheet. At the bottom left corner of the screen there is a Special addition/deduction items section. Open the menu and choose "Employee Loan" and enter the amount under Rate.
You can also write a manual paycheck. To do so, choose the Employee, set the Status to Mark as already printed and enter a Check number. Choose "Employee Loan" from the Addition/Deduction drop-down list and enter the amount under Rate.
To setup the Deduction for the employee go to Employees → Enter Employees and double-click on an employee, then click the Payroll tab. Using the drop-down list in the Addition/Deduction section, choose the "Employee Receivables" deduction, enter the amount (as a negative number) you want to deduct from each check under Rate and the amount of the loan under Limit.
In the example below $20 will be withheld from the next check and the limit will change to $180.00. The deduction will continue until the limit reaches zero. If the employee adds to his loan balance make note of the limit amount and add the amount of the new loan to the current limit. In the example below if the employee borrowed another $50.00 we would change the Limit amount to 250.00.
Running the report – To see the transactions go to Employees → Reports → Payroll Liability Report.
Set the Type to By Employee, choose None as the Reporting Type, enter an employee number to run a report for just that employee or leave blank for all, and enter your Employee Receivables GL account number in the Account field. This report will show you the loan amount, as well as the total deducted for all pay periods and the balance.
You can also view an employee's balance from the Employee Setup Screen under Additions/Deductions – Limit should coincide with your payroll liability report.


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Although Walter took the secret of this mission to his death, these survivors tell his story and reflect on his ability to carry on when others gave up. They leave us to question our own moral code and what each of us might do when faced with such choiceless choices.


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Amenities also create personal and economic value. People often desire to live near parks and schools as well as shopping, dining, entertainment and recreational opportunities.
We are fortunate to live in an area where so many amenities are planned to meet the growing needs of a rapidly developing community. Recent news stories demonstrate the number of amenities being added or planned here. A few of these assets are government facilities, some are private-public projects and many are simply businesses meeting the needs of the marketplace.
Ready for a show? Plans for a new luxury movie theater at Coastland Center mall in Naples are closer to showtime with approval of the redevelopment project this week by the city's Design Review Board. New movies are returning to the old mall to replace the Sears store and automotive center that closed last fall. The state-of-the-art CMX Cinébistro will feature chef-prepared American bistro cuisine with fine wines and signature cocktails.
Without fanfare, the renovated Hollywood 20 theaters relaunched Sunday in North Naples. Regal Cinemas used Hurricane Irma as an excuse to revamp the decades-old business inside and out, bringing IMAX and 4DX movie experiences to Collier County.
Stretching its legs, Gulfshore Playhouse's plans for new downtown Naples venue are advancing. A 45,000-square-foot theater is planned on Goodlette-Frank Road.
Meanwhile, the first area Dave & Buster's launched 40,000 square feet of video games, food and more family-oriented fun this week in a redeveloped retail area of Bell Tower Shops in south Fort Myers.
Believe it or not, Collier County is exploring possibilities to make its beaches wider and taller. How's that for growth and development?
Naples just opened part of Baker Park along the Gordon River after years of planning and construction. Another community green space that has been years in the making – Big Corkscrew Island Regional Park in Golden Gate Estates – got a pass recently from the county's sales tax advisory board.
Tipping the upscale, TwinEagles celebrates its golf-centric lifestyle with two championship courses and a remodeled Naples Yacht Club has expanded to stay in shipshape.
We've only scratched the surface of local amenities this week.
In future newsletters, we'll look at other projects such as Collier County's redevelopment plans proposed for Golden Gate aimed to attract new businesses and make a stretch of Golden Gate Parkway more walkable.
Have a local question? The longtime popular "In the Know" column is expanding with an additional answer man. Beginning next week, David Dorsey's weekly columns in the News-Press will focus on development in the northern parts of Southwest Florida, including Fort Myers and Cape Coral. Email Dave your local questions at intheknow@news-press.com.
Tim Aten will keep answering questions in the Naples Daily News about what's going on in the southern part of Southwest Florida. Email Tim your local questions at intheknow@naplesnews.com.
If you're not already a subscriber to read this premium content, you'll want to sign up so that you don't miss a thing. Here are some special offers so that you can be among the first to know what's going on in our ever-expanding community.
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Blog > Knowledge Management Fund > ...
Activation of the Crime of Aggression: African States the missing link!
by James Nwayo
Last year, the 17th July 2018, twenty (20) years since the adoption of the Rome Statute – the multilateral treaty that established the International Criminal Court ('the Court'), marked another milestone towards the recognition of the role that legalism can play in international relations. The crime of aggression was activated under the jurisdiction of the Court; hence the Court can prosecute the crimes of genocide, crimes against humanity, war crimes, and now aggression. That is, the Court can now hold individual political leaders accountable for committing the crime of aggression. The activation strengthened the international relations principle that waging wars of aggression is not a sovereign right but an international crime punishable in a court of law. Back in 1998, at the Rome Conference, where the Rome Statute was negotiated and adopted, the crime of aggression had been listed in the Rome Statute among the crimes under the jurisdiction Court.[1] However since the delegates at that time could not agree on a definition of aggression, the Court could not immediately exercise its jurisdiction over the crime. It had to wait until a provision was adopted defining the crime and setting the necessary pre-conditions to enable the Court to exercise jurisdiction over aggression.
This was achieved during the Review Conference of the Rome Statute, held in Kampala, Uganda in June 2010. In Kampala, the States Parties, those states that had ratified the Rome Statute, reached a consensual decision and adopted Amendments to the Rome Statute of the International Criminal Court on the crime of aggression.[2] The amendments provided a definition of aggression and also the pre-conditions to enable the Court to exercise its jurisdiction over the crime. Still there were steps to be followed for the activation of the amendments. First, the amendments required thirty (30) ratifications by States Parties. Second, and after the threshold of thirty ratifications is met, there was a requirement that after the 1st of January 2017 a decision was adopted by the majority of States Parties, adopting the amendments to the Statute. On the 26th of June 2016, The State of Palestine became the thirtieth State Party to ratify the amendments when it deposited its instruments of ratification. This paved the way for the Assembly of States Parties on 14th December 2017 to adopt a Resolution ICC-ACP/16/Res.5 activating the jurisdiction of the Court over the crime of aggression. In the resolution, it was decided 'to activate the Court's jurisdiction over the crime of aggression as of 17 July 2018'.
The last time that individual criminal responsibility had been applied to hold political leaders accountable for launching wars of aggression was at the Nuremburg Trials after the Second World War. In the London Charter of the International Military Tribunal the crime of aggression was codified under Article 6 as a 'Crime Against Peace'. Since then, the possibilities of prosecuting political leaders for waging wars of aggression has been like a mirage in the desert. Political leaders of militarily and perhaps economically powerful states have willy-nilly committed crimes of aggression with impunity. In other words, international order has been based more on the law of might rather than the power of international law. It would be natural to assume that the protective shield offered by the activation of the crime of aggression to militarily and economically weak states, many in Africa, would be enough to attract African States Parties to the Rome Statute, and to ratify the 2010 amendments in large numbers. It is therefore curious that Botswana is the only African State Party out of a possible 34 that ratified the amendments. More so, the statements by African States after the adoption of Resolution ICC-ASP/16/Res.5 activating the jurisdiction of the Court over the crime of aggression were tepid when compared to those of other States Parties. The delegate from Madagascar stated that his country had not ratified the Kampala amendments and was still conducting national consultations. This was before he emphasised Madagascar's attachment to paragraph 5 of article 121.[3] Paragraph 5 provides that any amendments to articles 5,6,7 and 8, pertaining to the Court's subject-matter (ratione materiae) jurisdiction, where only applicable to the States Parties that ratified the relevant amendments. A similar position was adopted by Nigeria. Uganda appeared to have been more excited by the fact that the origins of the amendments were in Uganda, as its delegate made no reference to if or when Uganda was planning to ratify the amendments.[4]
The conundrum is that during the drafting process of the Rome Statute in the late 1990s, African States' common position was that the Court's subject-matter (ratione materiae) jurisdiction should include the crime of aggression. This position was first articulated in September 1997 in the Common Statement of SADC Ministers of Justice/ Attorneys-General following their meeting in Pretoria, South Africa. In paragraph three (3) of The Common Statement of the Southern Africa Development Community (SADC) member states, stated that they;
Strongly believe that the Court must have inherent jurisdiction of the core crimes of genocide, crimes against humanity, war crimes in international and non-international armed conflict and aggression;[5] During the Rome Conference, African delegates spoke in favour of the inclusion of the crime of aggression under the jurisdiction of the Court. Mr. Sayed Kassem El Masry, Advisor to Egypt's Minister for Foreign Affairs described aggression as 'the basis of all crimes.'He even proposed that both the United Nations General Assembly (UNGA) and the United Nations Security Council (UNSC) be given legal authority to determine the existence of aggression. This was a departure from the status quo where such authority is the limited to the UNSC.
The initial support by African States for the inclusion of the crime of aggression under the jurisdiction of the Rome Statute could be interpreted from the fact that they had directly experienced it or witnessed how fellow African states had struggled against aggressive tendencies of the military and economically powerful states. Although much of the aggression was from the former colonial powers, as was the experience of Congo after independence from Belgium, other situations of aggression were solely between African states themselves. In Congo, Patrice Lumumba and Joseph Kasavubu described, probably accurately, Belgium's intervention in Congo's internal affairs as 'external aggression' in a letter dated 13 July 1960 to the United Nations Secretary-General. In 1986, the United States launched Operation El Dorado Canyon against Libya in the name of self-defence. However, the then Organisation of Africa Unity denounced the raid 'as not only a dangerous precedent but… a contemptuous and condemnable act in violation of the principles of international law.' African states such as Ethiopia and Chad also experienced aggression from their neighbouring states. For instance in 1977, Somalia, in pursuance of its irredentist foreign policy, invaded Ethiopia in an act which could be described as aggression. It is a truism that in general Africa has experienced a fraction of inter-state conflicts in comparison to intra-state conflicts. Still, that does not rule out that in future inter-state conflicts fueled by irredentist ideologies might resurface and possibly increase as more African States may choose to pursue nationalistic foreign policy and abandon the ideals of pan-Africanism and regional solidarity. The fissures are already there to see, mainly in relations to border disputes especially where valuable resources such as oil and gas are discovered. This is the case between Malawi and Tanzania, who are members of the SADC. The two countries have had diplomatic tensions over the territorial border of Lake Malawi, where gas deposits were discovered. In 2019, Kenya and Somalia almost had a complete diplomatic fallout over the alleged auctioning of oil and gas blocks along the disputed maritime border. One would have thought that a combination of the historical realities and the emerging internal and external threats of wars of aggression would be motivation enough for African states to ratify the amendments to the Rome Statute. It is regrettable that this has not been the case.
Perhaps the dispiritedness being demonstrated by African States Parties is a reflection of their general dissatisfaction with the entire edifice of the Court. The African Union has been categorical of its opposition to the manner in which the Court has been going about its business. The general sentiment has been that, arguably, the Court is only hunting African personalities for investigation and prosecution while ignoring personalities from militarily and economically powerful states such as the United States and the United Kingdom for the crimes committed in Afghanistan and Iraq. Both the disciples and the high priests of the Court would argue that this view-point is flawed, considering that some African States invited the Office of the Prosecutor to open investigations into their territories through 'self referral'. Still, the seemingly unconverted, suspect that the Office of the Prosecutor solicited such referrals. The difficulty with this argument is that it presupposes that African States lack the ability to make their own independent evaluation of what is best for them and their foreign policy decisions. In any case, such debates are beyond the focus of this post.
Another and perhaps more palpable argument is that African States continue to be wary of the role assigned to the United Nations Security Council in triggering the jurisdiction of the Court vis-à-vis the crime of aggression. Already African States have been consistent in their opposition towards the United Nations Security Council being given any substantive role in the operations of the Court. They had misgivings that such a role would inject raw-realist politics into the functioning of a judicial institution. Their concerns were prophetic. The United Nations Security Council has turned international criminal justice, which was intended to be a 'global public good' into a 'club good', preserved only for states such as Sudan and Libya while Syria is excludable. In addition, and as expressed by Mr. Alhaji Abdullahi Ibrahim,Minister for Justice and Attorney General of Nigeria, when he said that his delegation had:
…a reservation about the proposed role of the Security Council. While there should be a relationship between the United Nations and the Court under an agreement, he was opposed to conferring on the Council the exclusive right to determine when aggression was committed and to refer such cases to the Court. The Court should not be encumbered at the outset by avoidable political influences. The power of the Council under Chapter VII of the Charter of the United Nations should not extend to the Court.
In other words, it seems African States welcome the criminalization of acts of aggression as long as the United Nations Security Council is taken out of the equation. This possibly explains why the crime of aggression is also included in the African Union's Protocol on Amendments to the Protocol on the Statute of the African Court of Justice and Human Rights. However, the Protocol is finding it hard to attract the required ratification for it to enter into force. This raises questions around African States' seriousness in providing viable alternatives to the existing "hegemonic institutions." The risk, though, is that by neither ratifying the amendments to the Rome Statute nor establishing a credible and effective regional criminal justice mechanism, African States remain unprotected from aggression from both their neighbors or global superpowers. Now is the time to act to add the missing link – as the proverb says – "a stitch in time saves nine."
[1] UN Doc A/CONF.183/13 (Vol.II) United Nations Diplomatic Conference of Plenipontaries on the Establishment of an International Criminal Court, Rome, 15 June -17 July 1998, Official Records Volume II, Summary records of the plenary meetings and of the meetings of the Committee of the Whole
[2] Resolution RC/Res.6 of the Review Conference of the Rome Statute, Annex 1 Amendments to the Rome Statute of the International Criminal Court on the Crime of Aggression, 11 June 2010
[3] ICC-ASP/16/20Assembly of States Parties to the Rome Statute of the International Criminal Court Sixteenth session
New York, 4-14 December 2017 Official Records Volume I at p.85
[5] Sivu Maqungo, 'The establishment of the International Criminal Court: SADC' participation in the negotiations' (2000) 9 African Security Review 42-53
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The air conditioner is one of the most common appliances used in all kinds of buildings nowadays. Air conditioners range from smaller window units to full-sized tower systems that can cool down larger spaces quickly. However, you should understand that there are a series of different components that are designed to work properly within the air conditioner. From time to time, you will need to carry out repairs and maintenance to keep your air conditioner running smoothly. Failure to do so can lead to serious damage and can also affect your air conditioner. AC system repair is a common service offered by many companies that specialize in repairing HVAC units as well. Here are a few things that you should know about carrying out AC system repair.
In order to isolate the source of the damage and get repair work done on the air conditioner, you will first need to contact a local company such as Air Source Air Conditioning. The company will send over a team to your place in order to check both the inner and the outer units and then determine the cause of the problem. Sometimes, it's an issue with the electrical circuitry, while in other cases, the damage might be caused by an internal malfunction.
Before you give your air conditioning unit to any company for repairs, you have to find out how much it will cost. Make sure that you ask the company to give you an estimate for AC system repair in Oahu before you give the conditioner to them. In most cases, the company will need to remove the air conditioner and repair it at their workshop before bringing it back and fixing it in place. It's best if you take action quickly to prevent the air conditioner from sustaining more damage.


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cell death & disease
Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies
Jessica Rosati ORCID: orcid.org/0000-0002-4483-386X1,
Daniela Ferrari2,
Filomena Altieri1,
Silvia Tardivo3,
Claudia Ricciolini4,
Caterina Fusilli ORCID: orcid.org/0000-0001-7497-06575,
Cristina Zalfa2,
Daniela C. Profico6,
Francesca Pinos2,
Laura Bernardini7,
Barbara Torres7,
Isabella Manni8,
Giulia Piaggio ORCID: orcid.org/0000-0003-2114-18928,
Elena Binda9,
Massimiliano Copetti10,
Giuseppe Lamorte7,
Tommaso Mazza ORCID: orcid.org/0000-0003-0434-85335,
Massimo Carella7,
Maurizio Gelati2,4,6,
Enza Maria Valente3,11,
Antonio Simeone12,13 &
Angelo L. Vescovi1,2,4
Cell Death & Disease volume 9, Article number: 937 (2018) Cite this article
Establishing specific cell lineages from human induced pluripotent stem cells (hiPSCs) is vital for cell therapy approaches in regenerative medicine, particularly for neurodegenerative disorders. While neural precursors have been induced from hiPSCs, the establishment of hiPSC-derived human neural stem cells (hiNSCs), with characteristics that match foetal hNSCs and abide by cGMP standards, thus allowing clinical applications, has not been described. We generated hiNSCs by a virus-free technique, whose properties recapitulate those of the clinical-grade hNSCs successfully used in an Amyotrophic Lateral Sclerosis (ALS) phase I clinical trial. Ex vivo, hiNSCs critically depend on exogenous mitogens for stable self-renewal and amplification and spontaneously differentiate into astrocytes, oligodendrocytes and neurons upon their removal. In the brain of immunodeficient mice, hiNSCs engraft and differentiate into neurons and glia, without tumour formation. These findings now warrant the establishment of clinical-grade, autologous and continuous hiNSC lines for clinical trials in neurological diseases such as Huntington's, Parkinson's and Alzheimer's, among others.
Cell therapy remains one of the most promising approaches for the treatment of neurological disorders. Recent observations of improved motor function in Parkinson's patients as elicited from transplanted mesencephalic dopaminergic neurons, suggest that the harnessing of the healing potential of these techniques may finally be within our reach1. However, many of the currently accessible cell systems present us with serious hurdles, pertaining to donor tissue procurement, heterogeneity, availability and related technical or ethical concerns2,3,4,5.
Many of these issues could be alleviated by the use of stem cells, whose inherent expansion ability and functional plasticity could respectively increase availability and trigger therapeutic actions, such as the replacement of dead cells, immunomodulation, anti-inflammatory, trophic and homeostatic activities6,7,8,9,10,11,12,13. For a systematic clinical use of neural stem cells (NSCs)14,15,16,17,18, manipulation systems and preparations must guarantee the broad availability of donor cells with reproducible cell behaviour and therapeutic effects through (1) expression of the full complement of stem cell functional characteristics and (2) stable and extensive self-renewal properties.
We have recently stated that stable human NSCs (hNSCs) can satisfy these requirements. Having obtained current good manufacturing practices (cGMP) certification for hNSCs from miscarriages, we have successfully used them in a phase I trial, with intraspinal transplantation in 18 ALS patients15. We are now focusing on resolving the concerns deriving from the use of allogeneic hNSCs and related immune suppression19. Since the establishment of autologous hNSCs is both impractical and, de facto, impossible, we have derived these cells from autologous human induced pluripotent stem cells (hiPSCs).
Recently, various types of central nervous system (CNS) precursors have been derived from hiPSCs20,21,22; nonetheless, evidence of systems for establishing bona fide, hiPSC-derived hNSCs endowed with the complete range of defining stem cell characteristics is negligible20. We describe a reproducible system to establish stable hiNSCs, whose properties recapitulate those of hNSCs. This takes place under conditions that avoid foreign DNA integration and that should allow for certification of the emerging hiNSCs according to cGMP guidelines and their potential use for autologous cell therapy.
Generation and characterisation of hiPSCs
We generated virus-free hiPSCs from human skin fibroblasts using a non-integrating, episomal-based reprogramming system, under feeder-free and xeno-free conditions suitable for obtaining cGMP certification23,24,25. Data are from three distinct lines: hiPSC#1, hiPSC#2 and hiPSC#3, from healthy, consenting adults26. hiPSCs displayed a typical human embryonic stem cell (hESC) morphology (Fig. 1a) and expressed OCT4 and TRA-1-60 (Fig. 1b and Suppl. Figure 1a). The endogenous expression (Fig. 1c), and the absence of exogenous expression (Fig. 1d) of the pluripotency markers LIN28, OCT4, KLF4, SOX2 and L-MYC were demonstrated through quantitative real-time PCR (qRT-PCR). As expected, hiPSC#1, hiPSC#2 and hiPSC#3 produced teratomas upon subcutaneous injection in immunodeficient mice (Fig. 1e, f and Suppl. Figure 1b–e). The karyotype of each hiPSC line (46, XX) was normal (>20 passages, Suppl. Figure 2a). Only one (out of three cellular lines) contained a minor copy number variation (CNV) produced by cell amplification, maintained in the neurospheres without further genome modifications65,64,64. hiPSCs were mycoplasma-free (Suppl. Figure 2b). Thus, these lines fulfilled criteria for identifying properly reprogrammed hiPSCs.
Fig. 1: Characterisation of adult skin fibroblast-derived hiPSC lines.
a Phase contrast of hiPSC#1, hiPSC#2 and hiPSC#3. b Immunofluorescence images showing expression of OCT4 (green) and TRA-1-60 (red) in hiPSCs lines. Nuclei are counterstained with DAPI (blue). c Histograms showing mRNA expression of OCT4, LIN28, L-MYC and SOX2 in hiPSCs with respect to non-nucleofected, wild-type fibroblasts (Fibrowt). Data are normalised on actin expression, are shown as log10 fold changes (f.c.) and represent the mean ± SEM of three experiments in duplicate. d Histograms showing the absence of exogenous genes expression after five–six passages of hiPSC amplification. Nucleofected fibroblasts (Fibronf) are used as positive control. Data are normalised on actin expression, are shown as log10 fold changes (f.c.) and represent the mean ± SEM of three experiments in duplicate. e Growth curves of hiPSC-derived teratomas. f Histological analysis of teratomas generated by hiPSCs after subcutaneous injection in immunodeficient mice. Representative hematoxylin-eosin images showing the presence of ectodermal derivatives (sebaceous gland for hiPSC#1 and hiPSC#2, neuroepithelial rosette with evident melanin deposits for hiPSC#3), mesodermal derivatives (cartilage for hiPSC#1 and hiPSC#3, adipose tissue for hiPSC#2) and endodermal derivatives (intestinal epithelium for hiPSC#1 and non-keratinised epithelial lining for hiPSC#2 and hiPSC#3). Scale bars: in a and b = 50 μm, in f = 20 μm for hiPSC#1 and 10 μm for hiPSC#2 and hiPSC#3
Generation of hiNSCs
We hypothesised that NSCs would likely appear throughout embryoid body (EB) differentiation and that by applying the selective culture conditions favouring the growth of hNSCs, stem cells might be isolated and expanded at the expense of other cell lineages, as shown with primary CNS tissue8,27. To optimise the protocol and timing of hiNSC generation and amplification, we tracked the expression of radial glial markers throughout differentiation of EBs. We used BLBP28,29, GLAST30,31, GFAP32 and PAX633 as markers of the neuralisation onset, for up to 8 weeks after triggering EB formation. Small subsets of EB cells acquired radial glial characteristics beginning as early as 2 weeks from EB induction (not shown). Yet, at this stage, efficient expansion of hiNSCs failed, most likely due to the small population size. Therefore, EBs were grown in KnockOut Serum Replacement (KSR) medium for 2 weeks under normoxia (Fig. 2a, i, ii) and switched to hypoxic conditions for 6 additional weeks (Fig. 2a, iii, iv). Several translucent, lightly stained protrusions emerging from dark EBs were observed (Fig. 2a, iv). After 8 weeks, KSR was replaced with neurosphere growth medium, routinely used to establish continuous, clinical-grade hNSCs from miscarriages8,15. EBs, mechanically dissociated after 10 days, produced round clusters similar to neurospheres, which were serially amplified every 10–15 days (Fig. 2a, v), giving rise to functionally stable, steadily expanding lines. The lines were banked through cryopreservation, displaying 60–70% vitality upon thawing. Karyotypes remained stable, mycoplasma absent (Suppl. Figure 3). The bona fide nature of the hiNSCs was assessed molecularly and functionally in comparison to GMP-grade hNSC produced according to European Medicines Agency standards (AIFA aM 54/2018) and used in phase I trials for ALS patients (NCT01640067) and for secondary progressive Multiple Sclerosis (MS) patients (NCT03282760).
Fig. 2: Generation and characterisation of hiNSCs.
a Schematic diagram illustrating the overall strategy to generate hiNSCs from hiPSCs. Phase contrast images illustrate the typical morphology of cells at each stage. b Bar graphs of qRT-PCR showing mRNA-expression profile of hiNSCs, hiPSCs and brain-derived hNSCs respect to Fibrowt. c Bar graphs of qRT-PCR showing mRNA-expression profile of EBs, hiNSCs and hNSCs respect to hiPSCs. d Bar graphs of qRT-PCR showing mRNA-expression profile of hiNSCs and hNSCs respect to hiPSCs. Each bar represents the mean ± SEM of three independent experiments in duplicate. Data are expressed as log10 fold changes (f.c.) respect to non-nucleofected fibroblasts (b, Fibrowt) or hiPSCs (c, d). Data are normalised on actin expression. Scale bars: a, i = 50 μm; a, ii–v = 100 μm
Molecular characterisation of hiNSCs
Our first step was to exclude the permanence of cells expressing hiPSC markers in our hiNSC cultures. Expression of hiPSC markers in hiNSC#1, hiNSC#2 and hiNSC#3 was compared to their parental hiPSCs and to hNSCs (Fig. 2b). Differently from hiPSCs, neither hiNSCs nor hNSCs expressed detectable levels of pluripotency markers, such as OCT4 and LIN28. Conversely, L-MYC and NSC-putative marker SOX2 were expressed by both hiNSCs and hNSCs.
To exclude the presence of non-neuro-ectodermal cells, we compared the expression of mesodermal, endodermal and neuroectodermal lineage markers of the three hiNSC lines to EBs and hNSCs. hiNSCs did not express EOMES, T, GATA4, FOXA2 and SOX17, but retained a high expression of the early neural antigens SOX1 and NES, similar to hNSCs (Fig. 2c). Radial glial markers were consistently expressed in both hiNSCs and hNSCs throughout serial subculturing, confirming their similarity (Fig. 2d).
In conclusion, our hiNSCs were selectively positive only for neuro-ectodermal markers. These results suggest that the application of the neurosphere selection method enriched/amplified exclusively neural progenitors possessing hNSC molecular characteristics.
Functional in vitro and in vivo characterisation and safe expansion of hiNSCs
We then compared our hiNSCs to clinical-grade hNSCs using the neurosphere assay27,34,35,36, in order to verify whether our hiNSCs possessed the ex vivo functional characteristics of bona fide NSCs—extensive self-renewal and multipotency in particular—that are epitomised in native hNSCs8,37.
Stem cell properties of hiNSCs
When plated under the stringent conditions of the neurosphere assay, hNSCs form the typical neurospheres that we have also consistently observed in our hiNSC cultures (Fig. 2a, v). Moreover, if the perpetuation of the culture is factually sustained by true hNSCs8,15, hNSCs expand to large numbers, stably retaining their growth curve slopes over extensive passages8.
All three hiNSCs displayed this behaviour, though at a lower expansion rate than hNSCs (twofold versus fivefold every 10–15 days for over 15 passages (Fig. 3a). Starting from as low as 250 000 hiNSCs, the estimated cell number that can be generated exceeds 4 × 109, showing that hiNSCs possess the expected extensive self-renewal properties that also make them appropriate for cGMP certification. The protocol was then tested on additional hiNSCs from healthy and affected individuals38,39,40, demonstrating similar growth curves to hNSCs, with minimum variabilities due to genetic background.
Fig. 3: Stem cell properties of hiNSCs.
a Growth curves of hiNSC lines (passages 10–15) compared to brain-derived hNSCs in neurosphere growth medium. Each value point of the curve is the mean ± SEM of three independent experiments. b Histograms showing the quantification of i TUBB3 + neurons and ii GFAP + astrocytes out of total nuclei, after in vitro differentiation of hiNSC and hNSC lines. Each bar represents the value of three independent experiments ± SEM. Only the statistical differences between each single hiNSC line with respect to the hNSCs are shown. c Representative fluorescent images showing hiNSC-derived neurons (TUBB3, red, i–iv), astrocytes (GFAP, green, i–iv) and oligodendrocytes (GALC, red, v) at days i 10, ii 17, iii 24 and iv 31 upon differentiation. d Expression of i GLUTA (green) and ii GABA (green) in hiNSC and brain-derived hNSC progeny, after 24 days of in vitro differentiation. Scale bars: 50 μm
We then assessed if our hiNSCs displayed multipotency, i.e. the ability to differentiate into cells of the three main CNS lineages. hiNSCs and hNSCs were differentiated in parallel and detection of neuronal class III β-tubulinIII (TUBB3; neurons), glial fibrillary acidic protein (GFAP; astroglia) and GALC (oligodendroglia) was analysed at 10, 17, 24 and 31 days in vitro (DIV) (Fig. 3b and Suppl. Figure 4a).
At 10 DIV, immature neurons were detected, which matured within 17 DIV, generating a dense network of long-branched TUBB3-positive processes with varicosities up to 31 DIV (Fig. 3c, i–iv and Suppl. Figure 4b), which also expressed the late-stage neuronal marker MAP2 (Suppl. Figure 4c). We further observed progressive morphological changes during GFAP-positive astrocyte differentiation (Fig. 3c, i–iv and Suppl. Figure 4b). GALC-positive oligodendrocytes were detected at all time intervals for all hiNSCs (Fig. 3c, v and Suppl. Figure 5a).
The quantitative analysis (Fig. 3b, i, ii) showed comparable patterns of differentiation for all hiNSCs. At 10 DIV the percentage of TUBB3+ was 12.4% ± 1%, 13.18% ± 1.7% and 17.60% ± 3.03% for hiNSC#1, #2 and #3, respectively, and remained generally stable for up to 31 DIV (10.88% ± 1.1%, 11.23% ± 2.3% and 14.66% ± 1.8% for hiNSC#1, #2 and #3). The percentages of GFAP+ at 10 DIV were also comparable for hiNSCs (57.62% ± 3.6%, 47.86% ± 5.3% and 56.82% ± 10.10%, for hiNSC#1, #2 and #3) and slowly decreased over time (40.5% ± 1.1%, 26.58% ± 4.2% and 32.46% ± 2.2% at 31 DIV for hiNSC#1, #2 and #3). The number of GALC-positive cells was stable for up to 31 days (8.53% ± 0.78%, 12.39 ± 1.33%, 6.56 ± 2.9% for hiNSC#1, #2 and #3) (Fig. 3c, v and Suppl. Figure 5a). Considering that the total neural cell percentage at 31 DIV was circa 60%, we stained for NESTIN, confirming that a part of the population had not yet managed to progress in its differentiation (Suppl. Figure 5b).
The qualitative differentiation profiles of hNSCs and hiNSCs were similar, although quantitative analyses showed significant differences, (Fig. 3b, i, ii).
An initial analysis of the neurotransmitter phenotypes generated by hiNSCs showed that as seen previously for hNSCs8, a small fraction of these were partly glutamatergic (4.90% ± 0.8%, 6.03% ± 2.1% and 1.98% ± 0.3% for hiNSC#1, #2 and #3, GLUTA+, Fig. 3d, i and Suppl. Figure 4e) and partly gabaergic (7.92% ± 1.32%, 8.83% ± 1.8% and 7.13% ± 1.0% for hiNSC#1, #2 and #3, GABA+, Fig. 3d, ii and Suppl. Figure 4d).
Safety considerations: evaluation of proliferation activity
To exclude the possibility that hiNSCs might acquire uncontrolled growth capacity, prodromal to cell transformation and tumorigenicity, we verified that hiNSC retained a permanent cellular mitogen dependence15, a key functional test for cGMP hNSCs. When hiNSCs were cultured without growth factors, a progressive loss of proliferation capacity was observed, with the number of viable cells dropping to zero in five passages or fewer, as in hNSCs (Fig. 4a).
Fig. 4: Safe expansion of hiNSC lines.
a Growth factor-deprivation curves demonstrating the growth factors dependence of hiNSC lines. b qRT-PCR showing downregulation of hTERT mRNA expression in hiNSCs respect to hiPSCs. Each bar represents the mean ± SEM of three independent experiments for each clone (hiPSCs and hiNSCs) in duplicate. Data are expressed as log10 fold changes (f.c.) respect to non-nucleofected fibroblasts (Fibrowt) and are normalised on actin expression. c Comparison between the growth curves (i) and their slope (ii) of hiNSC (n = 15), respect to brain-derived hNSCs (n = 32) and hGBM (n = 12)
We evaluated hTERT expression, a hallmark of transformed brain stem cells41, absent in hNSCs. We found no expression of hTERT in the hiPSC-parental fibroblasts and, as expected, a high expression in hiPSCs, as a consequence of their successful reprogramming (Fig. 4b). Notably, telomerase expression became undetectable in hiPSC-derived hiNSCs, as in the hNSCs used as external controls (Fig. 4b).
These findings were confirmed through a statistical analysis comparing the kinetic growth slopes of three cell groups: (1) hiPSC-derived hiNSCs, n = 15; (2) hNSCs, used in a clinical phase I trial, n = 32; and (3) cancer stem cell lines derived from human glioblastoma (hGBM) specimens41, n = 12. hiNSCs showed slopes similar to hNSCs (p = 0.815) while, as expected, their growth was slower than hGBM cell lines (p < 0.0001) (Fig. 4c, i, ii).
Evaluation of engraftment and lack of tumorigenic ability of hiNSCs in immunodeficient mice
The hiNSC growth characteristics observed above support future applications in clinical settings. hiNSCs were implanted into immunodeficient athymic mice striatum (300 000 cells/animal, n = 11) and their growth was compared to animals receiving the same number of cGMP hNSCs (n = 10) (negative controls) and hGBM cells (n = 3, 150 000 cells/animal) (positive controls).
For the entire duration of the study (6 months), the animals receiving either hiNSCs or hNSCs did not present with clinical symptoms emerging from intraparenchymal cell overgrowth, i.e. behavioural anomalies, hydrocephalia, weight loss or ataxia. Brain haematoxilyn/eosin staining showed a normal cytoarchitecture of the striatal parenchyma even in the proximity of the injection site (needle track, Fig. 5a, i, ii) and confirmed the lack of exacerbated proliferation, as opposed to the large tumoral masses observed in mice receiving hGBM (Fig. 5a, iii). Furthermore, the immunohistochemistry showed that hiNSCs successfully engrafted, as shown by human-specific nuclei (huN) expression, migrating from the injection site towards the corpus callosum and cortical regions (Fig. 5b, i), similarly to hNSC behaviour (Fig. 5b, ii). Quantitative analysis of cell migration and survival (Fig. 5c, i, ii) also confirmed similar engraftment abilities between hiNSCs and hNSCs. We observed that hiNSCs seemed to be more prone to migrate from the injection site when compared to hNSCs (antero-posterior graft extension: 5.23 ± 0.55 mm for hiNSCs and 3.84 ± 0.44 mm for hNSCs, p < 0.05 (Fig. 5c, i). The total number of viable engrafted cells was 38.39 ± 9.29% for hiNPSCs and 20.68 ± 11.29% for hNSCs, (Fig. 5c, ii). Of note, the percentage of Ki67+ in hiNSCs (1.56 ± 0.44%) was lower than in hNSCs (4.21 ± 0.63%, p < 0.05) (Fig. 5c, iii; d, i).
Fig. 5: Engraftment and non-tumorigenicity of hiNSCs upon transplantation into the brain.
a Haematoxilyn-eosin stain of ipsilateral hemisphere of animals transplanted with i hiNCSs (n = 11), ii hNSCs (n = 10) and iii hGBM cells (n = 3). b Brain map showing the distribution of transplanted i hiNSCs and ii hNSCs (huN+, green) throughout the brain. In the magnifications are shown the brain regions evaluated for quantification analysis: cortex, striatum, corpus callosum, subventricular zone (not shown) and injection site. c Quantification of i migration, ii survival ability and iii of the relative percentages of neurons, astrocytes and oligodendrocytes generated by hiNSCs and hNSCs. d Confocal images showing the expression of i proliferation (KI67, red), ii neural (huNES, green), iii astroglial (GFAP, red), iv neuronal (TUBB3, red) and v oligodendroglial (MBP, red) markers for hiNSCs and hNSCs (huN+, green). Nuclei are shown by TO-PRO-3 staining (blue). Scale bar: a, b = 1000 µm, d = 75 µm, d, i, ii, v inserts = 17–19 µm, d, iv insert = 10 µm
Analysis of the differentiation pattern of transplanted hiNSCs
We evaluated the ability of hiNSCs to give rise to mature CNS lineages 6 months after intracerebral transplantation. Confocal analysis (Fig. 5d and Suppl Fig. 6) detected huN co-localisation with markers expressed by immature CNS progenitors (NES), astrocytes (GFAP), neurons (Doublecortin (DCX) and TUBB3) and oligodendrocytes (myelin basic protein; MBP), confirming the multilineage differentiation capacity of our hiNSCs, again similar to that of hNSCs. Quantitative analysis (Fig. 5c, iii) was performed on brain slices in the proximity of the injection site, showing that a similar percentage of hiNSCs and hNSCs expressed NES (11.21 ± 1.25% for hiNSCs and 17.18 ± 0.87% for hNSCs, Fig. 5c, iii; d, ii) and GFAP, (21.22 ± 6.37% for hiNSCs and 31.93 ± 0.79% for hNSC, Fig. 5c, iii; d, iii and Suppl. Figure 6). Interestingly, hiNSCs were more prone to differentiate into the neuronal lineage than hNSCs and this feature was evident when comparing both the most immature fraction of huN+ neurons, i.e. migrating neuroblasts positive for DCX+ (19.46 ± 2.93% for hiNSCs and 11.26 ± 0.87% for hNSCs, p < 0.05, not shown) and the more mature TUBB3+ neurons (28.82 ± 3.77% for hiNSCs and 10.34 ± 1.89% for hNSCs, p < 0.01, Fig. 5c, iii; d, iv and Suppl. Figure 6). Finally, oligodendroglial huN+/MBP+ cells (Fig. 5d, v) were detected in similar percentages: 5.80 ± 0.56% for hiNSCs and 5.60 ± 0.60% for hNSCs (Fig. 5c, iii and Suppl. Figure 6).
We analysed the whole-genome expression profiles of hiNSCs compared to: (1) parental hiPSCs and IPS lines derived from public database GSE6135842; (2) the hNSCs described in this article and NSCs derived from public database GSE61358; and (3) three glioblastoma cell lines obtained from public database GSE72218. Principal component analysis (PCA) (Fig. 6a) showed that the cell lines clustered together according to cell type, demonstrating that the inter-type cell line differences were maintained with respect to their genetic background. hiNSCs clustered closest to hNSCs, while undifferentiated iPS formed a separate cluster, which was closer to the group of glioblastoma cell lines. The comparison between hiNSCs and parental hiPSCs showed that the genes specifically expressed by hiNSCs were related to movement (z-score = 1.702, p < 0.0001), cell proliferation (z-score = 0.833, p < 0.0001) and differentiation of CNS cells (z-score = 1.186, p < 0.0001), while genes implicated in cancer (typical of hiPSCs) were silenced (Fig. 6b). These results supported our "in vitro" and "in vivo" experiments and confirmed the non-tumorigenic properties of these cells. Moreover, differentially expressed genes in the hiNSC group were shown to be significantly implicated in the process of apoptosis of brain cells (z-score = 0.770, p = 0.0023) and quantity of neurons (z-score = 0.901, p < 0.0001), which were turned on in these cells, while genes implicated in cell proliferation were turned off, as opposed to the hNSC group (Fig. 6c). This result is in line with our experimental data that demonstrated that hiNSCs have a lower growth rate than hNSCs.
Fig. 6: Gene expression analysis.
a Principal component analysis showing the whole-genome expression profiles of hiNSCs (purple cloud) in comparison with: hiPSCs (light green cloud, including hiPSC#1, hiPSC#2, hiPSC#3 and IPS lines derived from the public database GSE61358), hNSCs (pink cloud, including the GMP-grade foetal hNSC line described in this article and data derived from the public database GSE61358) and three glioblastoma cell lines (yellow, orange and dark green clouds, obtained from the public database GSE72218). b Differential gene expression of hiNSCs versus their parental hiPSCs. c Differential gene expression of hiNSCs versus foetal hNSCs
Extensive preclinical studies have furthered the use of neural precursors in clinical trials4,35,43,44,45,46. Furthermore, over 100 patients have now received implantations of hNSCs in authorised clinical trials worldwide14,15,16,17,18,47,48,49.
Extending these initial approaches to a larger body of patients is critically dependent on the availability of suitable sources of donor cells, which must retain reproducible and predictable cellular characteristics over a timespan long enough to generate an adequate amount of cells. This guarantees both that the treatment will be available to many patients and that all of these will receive the same cellular therapeutic agent for as long as necessary.
This manuscript describes a protocol for establishing hiPSC-derived hiNSCs, whose virus-free generation and stem cell characteristics provide an ideal source of autologous brain cells amenable for cell therapies in humans, showing that it is possible to isolate cells possessing all expected characteristics of bona fide hNSCs from skin fibroblasts50,51,52.
Generating hiNSCs from hiPSCs as described here may have several significant advantages. First, this system allows for two sequential amplification steps, the first regarding hiPSC growth and the second at the stage of hiNSCs, rendering the number of cells that can be used in neurological disorders virtually unlimited. Second, genetic manipulation can be carried out at the hiPSCs stage, passed on to the hiNSCs, which could be transplanted into patients. Third, the generation of autologous hiNSCs circumvents the need for immunosuppression regimens. Finally, on the basis of the functional homology between hiNSCs and clinical-grade hNSCs, our hiNSCs will facilitate cGMP certification previously granted to hNSCs.
Like their foetal hNSC counterparts, our hiNSCs had a stable expansion rate, did not possess telomerase activity, remained strictly dependent on mitogens for their proliferation and expansion and when growth factors were withdrawn, promptly differentiated into neurons, astroglia and oligodendrocytes. Of note, hiNSC correctly executed the differentiation process, as it normally occurs in hNSCs, so that the appropriate, univocal segregation of single pan-lineage-specific markers occurred within distinct, single differentiated cells (Fig. 3c), as opposed to the promiscuous expression observed in GBM cells41. Along the initial 24 days of in vitro differentiation, the number of hiNSC-derived neurons increased linearly over time (as for hNSCs), with a reduction on the 31st DIV. Interestingly, in the mouse brain, where trophic factors were present, at 6 months from transplantation, the percentage of hiNSC-derived neurons was greater with respect to hNSCs. It is likely that the in vitro experimental conditions are not optimal for full survival after differentiation.
Previous approaches have so far employed morphogenetic molecules to induce iPSC neuralisation53,54,55,56,57. We hypothesised that during long-term hiPSC-derived EB differentiation, radial glial cells, which are responsible for adult neurogenesis in the subventricular zone of the mammalian brain58,59, would develop. Having observed that this did indeed occur within a subset of cells, we applied a positive cell selection through a chemically defined medium. This permitted the exclusive expansion of the hiNSCs without the further addition of fate specification molecules. Significantly, our cells do not need to be committed towards specific neural differentiation before being inserted into the human brain, unlike formerly published protocols60,61,62, but could in the future be applied as single undifferentiated hiNSCs, whose differentiation would occur spontaneously through stimulation by the natural environment in which they will have been transplanted.
Our hiNSC preparation technique requires 2 months, followed by 4–8 months for line amplification, a much longer time window than those previously described54,55,56,57. However, these protocols do not stably amplify hNSCs as derived from GMP-grade iPCs. Thus, the dual advantages of our protocol are that we can produce these cells without adding inducing molecules and that neural cells can be obtained in the large amounts required for several serial transplants.
During our procedure, we repeatedly encountered two different types of cells, which appeared morphologically and functionally identical up to the sixth passage in culture, when differences emerged: the first type fit the definition of Transit Amplifying Progenitor (TAPs), which after a short proliferation (maximum seven passages) stopped growing and subsequently died (Suppl. Figure 6d). The second type fit the definition of bona fide hNSCs, corresponding to our hiNSCs, which continued to grow and thrive up through 25 passages. This has been explained in the literature58,59,63, which underlines the significant differences between these two groups of precursors: (i) bona fide stem cells, whose self-renewal spans or even exceeds that of the organism and (ii) short-term self-renewing TAPs, which have an intrinsically restricted self-renewal ability and terminally differentiate after a limited number of cycles43,44. These two types of cells possess very different functional and molecular properties, which have relevant consequences on their potential therapeutic applications. hNSC in general, and the hiNSCs described here, will sustain a long-term stable expansion process suitable for certifiable clinical-grade cell drug products with standard properties, TAPs will not. Rigorous long-term experiments should be performed on NSC lines, as mistaking TAPs for hNSCs may engender confusion and create significant issues with standardisation and reproducibility of results in cell therapy.
We further confirmed the safety of our hiNSCs and their similarity to cGMP-grade hNSCs, observing that transplantation into the striatum of athymic nude mice (6 months) showed no clinical symptoms known to accompany cell overgrowth in the brain. Histological analyses confirmed that none of our hiNSCs established hyperplastic or neoplastic masses or formed the typical teratomas observed after implantation of their parental iPSCs. Corroborating the results obtained in animal models, the first patient treated with cGMP hNSCs, has had a follow-up of more than 6 years without evidences of abnormal cell proliferation64,65.
Future studies will allow us to expand this approach and to define the full spectrum of possibilities that our technique offers for producing different subtypes of hiNSCs and for obtaining autologous brain stem cells that are amenable for therapy in humans. In reference to these objectives, hiNSCs produced with our protocol are not only suitable for receiving certification for safe implantation into humans, but may contribute to resolving many of the practical, regulatory and immunological issues that still afflict the use of foetal hNSCs in clinical settings.
Production and characterisation of hiPSC lines
The hiPSCs used in this study were iPS#1, iPS#2 and iPS#3. The first two lines were from a 41-year-old female donor and the third from a female donor aged 51. The lines were established through virus-free reprogramming. Briefly, we nucleofected 3 × 105 fibroblasts with 3 μg of a combination of three episomal vectors (pCXLE-hOCT3/4-shp53—Addgene #27077, pCXLE-hSK—Addgene #27078 and pCXLE-hUL—Addgene #27080). Six days after nucleofection, cells were detached and re-seeded onto a matrigel layer (Corning) and cultured in NutristemXF medium (Biological Industries). Small hiPSC colonies became visible between 5 and 6 weeks after transfection. We obtained a reprogramming efficiency of about 0.005% and 0.004% respectively for patient 1 and patient 2, corresponding to 16 and 12 iPSC clones. Each hiPSC line exhibited typical hESC morphology and expressed standard pluripotency markers, detected through immunocytochemistry (anti-OCT4 (1:100—Life technologies) and anti-TRA-1-60 (1:100—Life Technologies)) and qRT-PCR (for primers list see Supplementary table 1). The hiPSC colonies were mechanically detached and amplified once a week. If spontaneously differentiated colonies appeared, manual removal was carried out. We also tested their potentiality to differentiate into three embryonic layers, both through EB and teratoma formation assays. For the generation of EBs, cells were re-suspended in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 20% KSR, 0.1 mM non essential amino acids (NEAA), 1 mM l-glutamine, 50 μM 2-mercaptoethanol, 50 U/mL penicillin and 50 mg/mL streptomycin. Fourteen days later, EBs were pelletted and RNAs were extracted for qRT-PCR analysis. For teratoma formation, approximately 3 × 106 dispase-treated hiPSCs, in 100 μL of Matrigel, were injected into the right flank of nude mice. After 1 month, tumours were collected for histological analysis to check for their in vivo differentiation capacity into derivatives of all three germ layers. Teratomas were included in paraffin; standard haematoxylin-eosin and Alcian Blue staining were performed. For immunohistochemistry the following antibodies were used: VIMENTIN (DAKO, M0725), S100 (DAKO, Z0311), biotinylated goat anti-rabbit (DAKO, E0432) and biotinylated goat anti-mouse (DAKO, E0433). Absence of mycoplasma contamination was verified by PCR analysis using N-Garde Mycoplasma PCR kit (EuroClone). For karyotyping, iPSCs were cultured in chamber slides (Thermo Fisher Scientific) coated with Matrigel (1:100) in Nutristem medium for 2–3 days. Cells were treated with a COLCEMID solution (Thermo Fisher Scientific) with a final dilution of 0.1 µg/mL for 60 min at 37 °C. Metaphases were obtained by adding a hypotonic solution (30 mM KCl in 10% foetal bovine serum (FBS)), followed by incubation at 37 °C for 6 min and by fixation using a cold, freshly made 3:1 ethanol:acetic acid solution. Karyotype analysis was carried out on GTG-banded metaphases (resolution 450–500). Fifteen metaphases were counted and three karyotypes analysed. Only clonal aberrations were considered: an identical structural alteration or the gain or loss of a chromosome had to appear contemporarily in at least two or three different cell colonies, as specified in international system of human cytogenetic nomenclature (ISCN) recommendations.
Derivation and maintenance of human NSC lines
Each hiPSC line was expanded to at least 70% confluency. Spontaneously differentiated colonies were manually removed via gentle scraping and hiPSCs were then detached using 1 mg/mL dispase (Sigma) in DMEM-F/12. After gentle scraping, cell aggregates were placed into Petri plates and cultured as cell suspension in NutristemXF medium. This medium was substituted after 3 days with KSR medium (DMEM/F12, 20% Knock-out Serum Replacement (Life Technologies), 2 mM l-glutamine, 0.1 mM beta-mercaptoethanol and 1% non-essential amino acids). We maintained the embryoid bodies for 53 days in KSR medium, putting them in hypoxic conditions (5% CO2 and 5% O2) after the first 2 weeks. The medium was changed twice a week. After 53 days we began the selection of neural precursor cells with serum-free neurosphere growth medium8. After 10 days, cells were collected, mechanically dissociated into single cells and replated at high density in the same medium. In this phase, a single cell produces a sphere that can be routinely split every 10–15 days: up to 20–30 times. The mycoplasma contamination test and karyotype analysis were performed with the same protocols as those used for iPS cells; the only difference, for karyotype analysis, was the plating of neurosphere cells, which were attached onto cultrex and grown in the neurosphere medium to amplify cells. CNV analysis was performed using an SNP-array platform (Cytoscan HD; Affymetrix, Santa Clara, CA), following the manufacturer's instructions. Labelled DNA was hybridised for 16–18 h at 49 °C in a GeneChip Hybridisation Oven 645 (Affymetrix). The chip was washed, stained in the GeneChip Fluidic Station 450, and scanned with a 3000 7 G (Affymetrix) scanner. Copy number analysis was performed using Affymetrix Chromosome Analysis Suite software (ChAS v3.0; Affymetrix). CNVs were filtered as follows: 25 markers and 500 kb of minimal size.
hiNSC cryopreservation protocol
Neurospheres were collected in a 15 mL tube and pelleted by centrifugation. The supernatant was discarded, the pellet was gently re-suspended in 1.5 mL of growth medium supplemented with 10% dimethylsulphoxide and transferred to a cryovial. The cryovial was placed into a freezing jar containing isopropyl alcohol, kept for at least 4 h at −80 °C and finally transferred to the liquid nitrogen tank.
To thaw hiNSCs, the cryovial was transferred in a 37 °C bath until thawed. Cells were transferred into a 15 mL tube containing 5 mL of growing medium and pelleted by centrifugation. The supernatant was discarded and cells gently re-suspended in growing medium and transferred to a cell culture flask to allow for further expansion36. Vitality of thawed cells was evaluated at the first amplification passage by counting viable cells out of the total cell number.
Neural differentiation
Neurospheres were mechanically dissociated to yield a single-cell suspension and transferred onto cultrex-coated glass coverslips at the density of 10 000 cells/coverslip in neurosphere growth medium consisting exclusively of fibroblast growth factor 2 (20 ng/mL). Cultures were shifted after 72 h to a mitogen-free medium containing 2% FBS (default differentiation protocol). Differentiated cells were cultured for up to 5 weeks to obtain a mixture of neural cells containing astrocytes, neurons and oligodendrocytes. Immunofluorescence analysis was performed at different stages of differentiation: 10, 17, 24 and 31 days after plating (see below).
The spontaneous differentiation of hiNSCs in our protocol was used exclusively to demonstrate their multipotency; in fact, the neurons and astrocytes were not intended for subsequent utilisation in humans.
Immunocytochemistry and immunohistochemistry
Cultures were fixed for 10 min in freshly buffered 4% paraformaldehyde at room temperature, followed by two 1× PBS washes. After blocking with 10% normal goat serum (NGS), the cultures were incubated overnight at 4 °C with the following antibodies: anti-TUBB3 (1:400—BioLegend); GFAP (1:200—Dako); GALC (1:200—Merk Millipore); anti-GLU; and anti-GABA. After rinsing with PBS, cultures were incubated with the following secondary antibodies: anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 555 (Invitrogen). Cultures were then stained with Hoecht 33342 (Invitrogen) or 49,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Microphotographs were taken, using a Nikon C2 fluorescence microscope and NIS Elements 1.49 software. Data are reported as percentages of labelled cells over the total number of nuclei ± SEM. Each value represents the average of at least three independent experiments.
RT and qRT-PCR
Total RNAs were isolated from fibroblasts, hiPSCs and hiNSCs of each patient using TRIzol reagent (Life Technologies), following the manufacturer's instructions. RNA quality was assessed by determining ultraviolet 260/280 absorbance ratios at Nanodrop 1000 (Thermo Scientific) and examining RNA size distribution on RNA 6000 Nano LabChips (Agilent Technologies), processed on the Agilent 2100 Bioanalyzer, using the total RNA electrophoresis programme. Only RNAs with a RNA integrity number ≥ 8 were used for subsequent analysis.
Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer's instructions, after digestion with DNAse I (Life Technologies).
qRT-PCR was performed using a 7900HT Fast Real-Time PCR system (Applied Biosystem). For each gene of interest, qRT-PCR was performed as follows: each RNA sample was tested in duplicate and B-ACTIN was used to normalise transcript abundance and calculations were performed with the 2^DeltaDeltaCt method. Statistical analyses were performed on at least three independent experiments.
Sybr green reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystem) with the following PCR programme: denaturation 95 °C for 10 min; amplification 95 °C for 10 s, 60 °C for 10 s, 72 °C for 30 s, all of which was repeated for 50 cycles; final elongation 72 °C for 7 min; final dissociation step 95 °C for 15 s, 60 °C for 15 s and 95 °C for 15 s. TaqMan reactions were carried out using TaqMan Universal PCR Master Mix (Applied Biosystem) and PCRs were performed, following the manufacturer's instructions.
Primers are listed in Supplementary Table 1.
Animal studies were approved by the Italian Ministry of Health Ethics Review Committee for Animal Experimentation, following protocol AUT. 651/2016 PR. Adult female Hsd Athymic Nude-Foxn1nu (Envigo) (15–20 g). hiNSCs were seeded (1 × 105 cells/cm2) in growth medium for 24 h. On the day of transplantation, cells were counted and re-suspended in Hank's balanced salt solution (medical) (density of 1 × 105 cells/μL). Mice striatum were unilaterally and stereotaxically (David Kopf Instruments, Tujunga, CA) injected with 3 μL of each cell suspension: hiNSCs and brain-derived hNSCs, 3 × 105 cells/mouse, n = 21; GBM cells: 150 000 cells/3 μL/animal, n = 3. Animals were analysed for 6 months (for hNSCs) and 3 months (for GBM). For the immunohistochemical analysis, mice were euthanized and transcardially perfused-fixed with 4% paraformaldehyde. Brains were post-fixed overnight, cryoprotected, frozen and coronally sectioned (20-μm thick) by cryostat. Sections were blocked with 10% NGS and 0.3% Triton X-100 for 90 min. Primary antibodies used: huN, NCL-KI67p (KI67, Novocastra), human NESTIN (NES) (R&D Systems, Minneapolis), GFAP (Dako Cytomation), TUBB3, MBP and DCX (Santa Cruz). Fluorescent secondary antibodies were labelled with Alexa Fluor 549 and 488 (Molecular Probes). DAPI (ROCHE) or TO-PRO3 Iodide (Molecular Probes) were used as nuclear markers. Labelled samples were analysed by fluorescence microscopy (Zeiss Axioplan 2 imaging) and by confocal microscopy (Leica DM IRE2). The survival rate of transplanted cells was evaluated by counting huN+ cells in serial brain sections (each 20 μm apart) spanning the graft area. The total number of surviving transplanted cells was calculated for the whole graft using the Abercrombie formula (Abercrombie, 1946). Data are presented as the average percentage of surviving cells over total transplanted cells (300 000). The antero-posterior migration was calculated by evaluating the distance between the most proximal and most caudal section containing huN+ cells. The evaluation of proliferating cells and neural phenotypes derived from transplanted cells was performed by calculating the percentage of huN+ cells co-expressing, respectively, Ki67, NES, GFAP, TUBB3, MBP and DCX out of the total huN+ cells in three serial sections of the transplanted animals (n = 8).
Bioinformatics analysis
The whole-transcriptome profiles of the herein described hiPSCs, hiNSCs and hNSCs were compared with those of publicly available data sets (GSE7221842 and GSE61358). These Gene Expression Omnibus data sets belong to the platform GPL17586, which contains data generated with Affymetrix Human Transcriptome Array 2.0 technology. In particular, in GSE72218, total RNA was extracted from both tumour tissues and three cell lines (U3020MG, U3047MG and U3065MG) intracranially transplanted into nonobese diabetic/severe combined immunodeficiency mice. The second cohort, GSE61358, concerns total RNAs extracted from hiPSCs and induced pluripotent stem cell-derived NSCs (iPS-derived neural precursor cell NPC). Our raw data, together with those of GSE72218 and GSE61358, were analysed through the Exploratory Grouping Analysis (EGA) available from the Transcriptome Analysis Console (TAC) 4.0 software. The first three principal components (PC) of the PCA of EGA were visualised through the rgl package of R software (ver 3.4.4). Differential expression analysis was performed using TAC (one-way analysis of variance (ANOVA)) and only genes with differential expression greater (absolute values) than twofolds were taken into consideration for further analyses. Functional enrichment analysis was performed by Ingenuity Pathway Analysis (IPA®) software (QIAGEN Inc.).
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This work was supported by Italian Ministry of Health, Ricerca Corrente 2014–2017 to A.L.V.; ERC Starting Grant 260888 to E.M.V., Association Revert Onlus, Fondazione Cellule Staminali.
Cellular Reprogramming Unit, IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Jessica Rosati, Filomena Altieri & Angelo L. Vescovi
Department of Biotechnology and Biosciences, University of Milan Bicocca, Piazza della Scienza, 220126, Milan, Italy
Daniela Ferrari, Cristina Zalfa, Francesca Pinos, Maurizio Gelati & Angelo L. Vescovi
Neurogenetics Unit, IRCCS Santa Lucia Foundation, Rome, Italy
Silvia Tardivo & Enza Maria Valente
Stem Cell Laboratory, Cell Factory e Biobank, Terni Hospital, Via Tristano di Joannuccio 1, 05100, Terni, Italy
Claudia Ricciolini, Maurizio Gelati & Angelo L. Vescovi
Bioinformatics Unit, IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Caterina Fusilli & Tommaso Mazza
Production Unit of Advanced Therapies (UPTA), Institute for Stem-Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Daniela C. Profico & Maurizio Gelati
Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Laura Bernardini, Barbara Torres, Giuseppe Lamorte & Massimo Carella
Department of Research, Diagnosis and Innovative Technologies, Regina Elena National Cancer Institute, Rome, Italy
Isabella Manni & Giulia Piaggio
Cancer Stem Cells Unit (ICS), Institute for Stem-Cell Biology, Regenerative Medicine and Innovative Therapies (ISBReMIT), IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Elena Binda
Biostatistic Unit, IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini, 71013, San Giovanni Rotondo, Foggia, Italy
Massimiliano Copetti
Department of Molecular Medicine, University of Pavia, Via Forlanini 14, 27100, Pavia, Italy
Enza Maria Valente
Institute of Genetics and Biophysics Adriano Buzzati Traverso, CNR, Via P. Castellino 111, 80131, Naples, Italy
Antonio Simeone
IRCSS Neuromed, 86077, Pozzilli, Isernia, Italy
Jessica Rosati
Filomena Altieri
Silvia Tardivo
Claudia Ricciolini
Caterina Fusilli
Cristina Zalfa
Daniela C. Profico
Francesca Pinos
Laura Bernardini
Barbara Torres
Isabella Manni
Giulia Piaggio
Giuseppe Lamorte
Tommaso Mazza
Massimo Carella
Maurizio Gelati
Angelo L. Vescovi
Correspondence to Jessica Rosati or Angelo L. Vescovi.
The authors declare that they have no conflict of interest.
The hiPSCs were derived from skin biopsies of two healthy human subjects who signed fully informed consent forms. All procedures performed in studies involving human participants were conducted in accordance with the standards of the Ethics Committee of the Institute "Casa Sollievo della Sofferenza". Human foetal brain tissue specimens, all derived from the forebrain, were routinely collected from foetuses that had undergone natural, spontaneous in utero death (miscarriage) at gestational ages beyond the eighth post-conceptional week. Tissue procurement was approved by the Ethics Committee of the Institute "Casa Sollievo della Sofferenza" and was possible exclusively after receiving the mother's informed written consent. Furthermore, specimen collection and medical procedures were in full accord with the Helsinki declaration (WMA Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects).
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Q: localhost:8080/abc/swagger-ui/index.html showing 404 not found after migrating from swagger2 to openApi3 Migrated to openApi3 SpringBoot project. But I'm not able to get swagger ui page while /v3/api-docs/ url working. But swagger url showing 404 not found. I'm also able to access /v3/api-docs/swagger-config/.
I'm using springdoc-openapi-ui dependency having version 1.6.11. I tried every possible way. For one time UI was displaying somehow but after restarting server it disappears. FYI, I've implemented SwaggerConfig.java and ConfigWebSecurity.java for ignoring url from spring security.In short i have implemented all those things which is mentioned for migration from swagger to openApi3 in official documentation.
FYI -
I have migrated 2 other applications by following same process. But for this app I'm facing this issue.
Thank in advance

A: what url are you trying for ui page ?
Try using localhost:YourPort/swagger-ui.html .

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